microscopy embryology workshop
TRANSCRIPT
Workshop30-31st May 2012
Research Instruments
Welcome & Introductions
Rob Watkins UK/IRE Business & Sales Manager
Martyn Selley UK/IRE Sales Manager
Martin Mankee International Service Manager
Directors:Bill, Justin & David
Day 1 MorningWednesday• DAY 1: 8.00 – 9.30 Reception
The office will be open and able to receive trainees for Tea /Coffee reception (for early arrivals!) til 9.30am Please arrive each day in advance of the scheduled start time so we can start promptly
Some attendees will have arranged for RI to pick them up from local accommodation - This will be carried out at this timeLocation: RI Head Office
• 9.30 – 10.15 – Directors and UK Managers welcomeIntroductions and reception with a tour of the facilities and transfer down to the training Suite at Site BLocation: Boardroom RI Head Office
• 10.15 – 10.30 – Introduction and Training aims for the practical sessionTraining aims and individual itinerary discussionFamiliarisation with the format and availability of the range of equipmentLocation: Training Suite Site B. The rest of the sessions will take place here
• 10.30 - 11.30 Session 1 Microscopy – Basics (Nikon, Olympus, Leica)Fundamentals of microscopy work and sample illumination
• 11.30 – 11.45 Short Break• 11.45 – 12.15 Session 2 Microscopy – IVF
Principles for Microscopy in the IVF application including proper set up of Condenser Illumination, Microscope and Optics
• 12.15 – 12.45 Fundamentals of Micromanipulation Integra and Manipulation techniques
• 12.45 – 13.45 Lunch – Off Site Location
Day 1 Afternoon• 13.45 – 15.45 Micromanipulation Continued
– Practical session on the use of micromanipulators - including but not exclusively:• Pipette selection and set-up• Quick set up guide of Pipettes on stage• Understanding the TDU• Understanding the PL30 and MPH• Overview of the HUD and Heated stages – Limitations and Benefits of ITO surfaces• Trouble shooting for common errors in set up in an IVF Lab
• 15.45 – 16.00 Short Break• 16.00 – 16.45 System and Pipette set up challenge
A fun recap on the practical learning's of the day’s study
• 16.45 – 17.00 Close of Day Key learning's and Transfers to accommodation
• 19.30 - Late Evening Events – Smart CasualDinner and Drinks in a seasonally appropriate venueDetails to be confirmed in the morning with the Students
Day 2 Morning
• Thursday• Day 2 • 8.30 – 9.00 Hotel Transfers to RI Training Suite• 9.00 – 10.00 Meeting Start - Manipulator Mania
A fun, practical challenge for two teams to enhance and develop both the understanding and use of the main assemblies and tools in micromanipulation learnt on day 1 and applied in these day’s sessions
• 10.00 – 11.00 Introduction to Advanced TechniquesBiopsy and IMSI use in IVF with the RI Saturn ACTIVE and RI IMSI
• 11.00 – 11.15 Short Break• 11.15 – 12.45 Manipulation and Biopsy I
Instruction on using laser assisted techniques and softwareOpen session and material available for a wide range of techniques either building on the previous days learning's or the start of new skills on more advances equipment
• 12.45 – 13.15 Lunch - On site catering
Day 2 Afternoon• 13.15 – 13.45 Manipulation and Biopsy II
Further hands on practical time and practice on the latest available ICSI/PGD and IMSI equipmentThroughout the sessions and during time when other equipment is taken up with other students instruction for use of the following environmental monitors will be available
• 13.45 – 1400 Short Break and Sugar• Cornwall’s best tradition to keep us going
• 14.00 – 15.15 Individual Development Session – The Final Session!Individual itinerary focus for students to cover any aspect of the two days as they and the trainer see fitThere will be almost 1-1 coverage of each aspect of the two days learning at each work areas with a wealth of RI trainers available for support
• 15.15 – 15.30 Close of Workshop and ThanksTransfer to accommodation or trains and departures
• 15.30 – Late After HoursThe RI UK team will be available in the training suite for a further hour for those staying onAnyone staying another night in Falmouth are welcome to join us again for an evening out
How is this session going to work?• Please Remember this is designed to be a practical workshop
throughout• There will be a little “Death by PowerPoint”• Although I need to show you something on the board, I believe in
“see one, do one” teaching methods • There is a reason that Microscopes do not appear too often in your
Embryology text books!Matchsticks are available!
Learning Objectives TodayToday you will:
Understand the fundamentals of the modern microscope used in the IVF process Undertake training of stereo and inverted microscopes and the contrast method for equipment you either currently have or are likely to be exposed to. Take away useful day to day operational and set up procedures of the Integra TI and associated hardware
An Industry Certificate will be provided. This will be sent to you from RI head office after the session.
ACE SECTION - MICROSCOPY• With the aim of a diagram describe the optical principles of a stereo
microscope• With the aid of a diagram describe the components of a compound
microscope• When are the following used
– i. Brightfield– ii Darkfield
• When might oil immersion be used and why• Using a diagram describe the principles of phase contrast, Hoffman and
Nomarski (DIC)• Use scopes in your lab• List procedure for cleaning • List the equipment in your lab and reason for use.
Microscopy for IVF
Brief BackgroundThe optical microscope, often referred to as the "light microscope", is a type of microscope which uses visible light and a system of lenses to magnify images of small samples.
Optical microscopes are the oldest design of microscope and were designed around 1600.
Basic optical microscopes can be very simple, although there are many complex designs which aim to improve resolution and sample contrast.
Historically optical microscopes were easy to develop and are popular because they use visible light so the sample can be directly observed by the eye.
Introduction to Our Limitations1. Physics – Its limitations on us in IVF
Any consideration of microscopy must begin with an understanding of how we perceive light and how light interacts with matter. Light has both a particle and a wave nature. For most of this discussion, it is the wave (sine wave) property of light which is of interest to us.
It also travels in straight lines.
• There are only 2 things that the eye can see. ???
The CONES respond to bright light and mediate high-resolution COLOUR vision during daylight illumination (also called photopic vision). Three types sensitive to (RGB) primary colour
The RODS are saturated at daylight levels. Rods respond to INTENSITY and mediate lower-resolution, monochromatic vision under very low levels of illumination (called scotopic vision).
2. Biology – Its limitations on us in IVF
Wavelength is the property we see as colour
The human eye sees (RGB) light in equal amounts as white light. All the other colours are generated by stimulation of the three different types of cones. Black is the absence of colour and / or intensity.
Coherence Coherent waves are all of the same frequency and all the waves are in phase (that is, all the waves are "up" at the
same point). Laser light is coherent.
A green light can be monochromatic but still be incoherent because the waves are in random phase.
Actually, an incoherent wave would have some dispersion, although it might be quite narrow, but it can't be too narrow, or it would be coherent for all intents and
purposes.
White light is incoherent both because the phase of the waves are random and because white light is made of
many different frequencies simultaneously.
Please bear in mind not all light is the same…
Wavelength
Colour always arises from light, there is no other source
All Colour super impose depending on the predominant wavelength palette
So we can only see our specimen if it has a different 1.COLOUR (wavelength) or2.INTENSITY (amplitude)
…than its background
…In IVF, our samples are very small and have no colour.
3. Intensity - Represented by Amplitude
How do we see objects that are not light sources themselves ?
• There are different processes, which happen when light meets physical matter. They are:
Reflection:- is a change in direction at an interface
Refraction :- is the change in direction of a wave due to a change in its speed
Diffraction:- is the bending of a wave around objects or the spreading after passing through a gap (slit)
Absorption:- the way by which the energy is taken up in matter
Colour Shift• A colour phenomenon – An everyday experience
– Clothes bought in the shop often look different when brought out into day light
This can be observed in the microscope if we focus on an object and increase the power slowly
The image observed will change from a yellow-red to a more bluish and then very bright image
THIS MEANS THAT WITH AN INCREASING POWER, THE INTENSITY OR AVAILABILITY OF DIFFERENT WAVELENGTHS (COLOUR) HAS BEEN CHANGED
Summary of Light in Our Workshops here at Research Instruments
• The light that reaches our eye consists of many different colours, and different light sources produce a mix of these
• Our vision gives us only a partial view of reality• For effective imaging the microscopist must be aware of the
complexity of the world of light• There is a variety of light sources, objectives and filters to
enable and assemble the microscope for a purpose• Several hundred years of development of optics have
managed to hide come of the complexity from the regular use
COFFEE!
Summary of Session One
• In this work shop we are using white light as you do in the lab• White light is made up of many different types of wavelength
(colours)• Light is seen by its colour and / or its intensity – in relation to
its background• We are limited by what our eyes can see, although the very
perception of an image is a remarkable process using complex structures that both catch ,process then interpret them
Light Microscope Schematic diagram
of a light microscope outfitted for
photomicrography
And what’s the difference between this and the invert ?
Jargon• Resolution- Resolution can be defined as the least distance between 2 points at
which they can still be recognized as 2 separate entities
• For the eye, this is 70 microns, when the object is 250mm away
• For light microscopy, this is 0.24 microns• Contrast-The phenomena that allows you to distinguish relevant information from
irrelevant. Either by colour or intensity
• Contrast and Resolution are inversely proportional
What is Resolution ? The ability to visually separate dots
Every specimen detail that is illuminated creates a so called diffraction pattern or airy disk.
When two details within the specimen are closely together we can only see them separated If the two central spots are not too close together and the airy disks themselves do not overlap
Resolution
Note: If the incident radiation contains several wavelengths, each wavelength deviates through a specific angle, the shorter the wavelength the less deviation and thus the better the resolution
• The smaller the Airy disks, the higher the resolution in an image. Objectives which have a higher numerical aperture produce smaller airy disks from the same specimen detail than low NA objectives
Resolution and Limit of Resolution
Limit of Resolution for a light microscope is 1000x Magnification
Wavelength versus Resolution
Wavelength(Nanometers)
Resolution(Micrometers)
360 0.19
400 0.21
450 0.24
500 0.26
550 0.29
600 0.32
650 0.34
700 0.37
Electron Microscopes – remember our limits…
• Benefits are that they do not use light but fire electrons that allow us to see detail smaller than wavelengths of light itself
• Therefore their Limit of Resolution (LOR)is much higher (A light microscopes LOR=1000x)
• Limit of resolution is near 1 million times therefore magnification of 350,000x is possible with Electron Microscopes - Significantly better than what we use
• TEMs – Transmission (2D) through the samples• SEMs – Scanning (3D) bounce off the samples• Cells have to be stationary and preparation and scanning actually kills denatures
them• We have to make a compromise with IVF samples
Jargon• Numerical Aperture- This indicates the resolving ability of an objective. Larger N.A.= Greater
resolution and also brighter fluorescence signal. However larger N.A.= less depth of field and shorter working distance. NA= nSinA, where n= refractive index of medium and A is the half angle at which light enters the objective.
NA
Resolution
Depth of Field
Shorter Working distance
1
0
NA = n Sin
Numerical Aperture
ImmersionMediaRefractiveIndex
Angle of theCone of illumination
Numerical Aperture N.A.
N.A. can never be higher than the refractive index of immersion mediaThe refractive index of the imaging medium is critical in determining the working numerical aperture of a microscope objective. A dramatic increase in numerical aperture is observed when the objective is designed to operate with an immersion medium such as oil, glycerin, or water between the front lens and the specimen cover glass
Jargon• Polarisation – The rotation and direction of the light wave expressed as its
orientation
• Working Distance- (W.D)The distance between the in focus specimen and the front lens of the objective
• Depth of Field– (D.O.F) Depth of field in a microscope is the area in front of and behind the specimen that will be in acceptable focus
• Field of View- (F.O.V) This is the area of the specimen in view down the eyepieces. It is dependant on the magnification and the Field number(F.N) of the eyepiece. F.N. of 22 indicates a F.O.V. diameter of 22mm when using a 1x objective
• Convolusion – Areas above and below focal plane causing glare, distortion and blurriness
Orientation and Polarisation(end-on view)
(end-on view)
(end-on view)
(end-on view)
Polarised light in Microscopy and Illumination Techniques
Huygens’ Principle and Diffraction Pattern
Numerical Aperture (NA) and Working Distance (WD)sample
WD WD WD
Resolution
Let’s not get too involved here…Far away… NA 0.2
ABDFGRGBNHYUKFLGMNJDEGFKGLWLKLIIURRM
Closer… NA 0.9
ABDFGRGBNHYUKFLGMNJDEGFKGLWLKLIIURRM
Jargon• Chromatic Aberration- When white light passes through a convex lens, the colours
split and focus at different points causing colour fringing - Objectives have additional elements to overcome this problem
• Spherical Aberration- When light passes through a material (glass/plastic) media
• Apochromatic objectives are fully corrected and Achromatic objectives are corrected for red/blue
• Plan objectives are designed, assuming a flat specimen, to provide a focused image across the whole field of view
Aberrations
BA
TS
C
Chromatic Aberration Coma
Spherical Aberration Astigmatism
On-Axis Off-Axis
Chromatic aberration
Objectives
Objective Lens Nomenclature
Red = 4xYellow = 10xGreen = 20xBlue = 40 x
Objective Lens Types
Types of Objectives / Heads• No designation – Dry Objective / Non Oil
• Infinity – High quality infinity corrected
• Oil Objectives (Oil – OI) NA>0.9 must be oil
• PLAN objectives (FN) Flat even focus across the Field of View (high quality)
High Mag. Objectives & Correction CollarCorrects for Spherical Aberration
Caused by variations in material thickness and type • ITO stages Tokai vs Standard• Different types of dish• NUNC four well vs Falcon circular..
Rotation either increases or decreases the distance to first objective lens
When we alter the light beam we affect the brightness. The more we do, the less light we have to push through and to see with, hence:Image Brightness: Numerical Aperture Magnification2
Correction Magnification Numerical Aperture F(trans)
Plan Achromat 10x 0.25 6.25
Plan Apo 10x 0.45 20.2
Plan Fluorite 20x 0.50 6.25
Plan Apo 20x 0.75 14.0
Plan Achromat 40x 0.65 2.64
Plan Apo 40x (oil) 1.30 11.0
Plan Fluorite 60x 0.85 2.01
Plan Apo 60x (oil) 1.40 5.4
Plan Apo 100x (oil) 1.40 1.96
Plan Apo 100x (oil) 1.45 2.10
Light-Gathering Power
Eyepieces• Typically 10 x sometimes 15x Magnification• These form an integral part of the magnification of the whole system• Separate to any camera imaging• Right eye is usually fixed and the left has a Dioptor adjuster• Pupil distance adjustment for user comfort
Head assembly and Prism split• 50/50 prism = 50% to eyepieces 50 % to camera (basic head)• Trinocular head has additional prism 100% to eyepieces or 20/80 split - usually only
important for Darkfield and Fluorescence
COFFEE!
LUNCH & DINNER(Weather Dependant)
• Dinner Tonight :– Choices
• Sea Food• Combination• Beach• Italian
LUNCH Menu choices on the board
• The job of the light based equipment (Light, polariser, condenser, HMC etc) is to present the light for the objective to do its work
• The objective is the key part and acts like our eye
• Our human vision is triggered to seethree dimensions and is well trained tointerpret structures if they are illuminatedmore or less from one point.
• We are limited by what we can see and the physics involved in the process
• Remember that a compromise is just that…
Visualisation Techniques
Visualisation Techniques Brightfield – Good for stained specimens
Darkfield – True colour against black background
Phase Contrast – Seeing live cells in plastic vessels
DIC – differential interference contrast Pseudo 3D of live cells in glass vessels only
HMC – Pseudo 3D of live cells in plastic vessels
Fluorescence – Sensitive method for localisation of chemical or physical changes (Tags)
Brightfield Phase Contrast DIC Nomarski
Visualisation Techniques
Brightfield Darkfield
Visualisation Techniques
What is Darkfield then....• Stars in the sky....
– We cant see the stars in daylight on a clear day yet we know they are there...
– We cant see dust the air in a room (most of the time!)• Yet if we shine a torch in a dark room we pick up the
dust in our vision because they diffract and/or reflect the light
• That is Darkfield or darkground illumination• Light is directed to the specimen in a way that no
direct light enters the objective
Bright field
Light path
IMAGE Objective Sample
Darkfield Cont.• Therefore we can see a particle or sample in front of a black
background even when the particle itself is too small to be resolved or does not show an appropriate contrast under bright field
IMAGE Objective Sample
Light path
Diagram of a dedicated Bright field – Dark field system
Oblique Illumination• The simplest way to achieve a contrast
method that results in a relief-like image (2D/3D)is by using oblique illumination
Structures within a specimen canbe identified and even though they areonly two dimensionally displayed theylook three dimensional.
Oblique IlluminationDirect Illumination
Limits of Oblique illumination
The contrast itself is produced by the complete thickness of the specimen and the resolution of the image is limited due to the oblique illumination
To overcome the limitations of oblique contrast, Hoffman, Nomarski Differential Interference Contrast (DIC) and others are commonly used for high resolving images. The benefit of this method is that the relief like image is only contrasted at the focus area (depth of field). The user can optically section a thicker specimen by changing the focus level.
Polariser in Microscopy
• Below the condenser, a circular polarizer is placed on the light exit port of the microscope. The rotation of this polarizer can control the effective width of the slit opening
The part of the slit controlled by the polarizer registers on the bright area of the modulator. As the polarizer is rotated, contrast can be varied for best effect. A very narrow slit produces images that are very high in contrast.
HMC
• Hoffman Modulation Contrast - Hoffman modulation contrast is an oblique illumination technique that enhances contrast in both stained and unstained specimens by detection of optical phase gradients.
Hoffman• The Hoffman Modulation Contrast system
is designed to increase visibility and contrast in unstained and living material by detecting optical gradients (or slopes) and converting them into variations of light intensity.
• An optical amplitude spatial filter, termed a "modulator" by Hoffman, is inserted on the back focal plane of an achromat or planachromat objective (although higher correction can also be used). Light intensity passing through this system varies above and below an average value, which by definition, is then said to be modulated
• Unlike the phase plate in phase contrast microscopy, the Hoffman modulator is designed not to alter the phase of light passing through any of the zones.
• When viewed under modulation contrast optics, transparent objects that are essentially invisible in ordinary brightfield microscopy take on an apparent three-dimensional appearance dictated by phase gradients
Principles of light path in Hoffman
Hoffman Modulation Contrast (HMC)Visualisation Techniques ii
Condenser• Above the stage, a condenser
with rotating turret is utilized to hold the remaining components of the Hoffman Modulation Contrast system.
• The turret condenser has a brightfield opening with an aperture iris diaphragm for regular brightfield microscopy.
• At each of the other turret openings, there is an off-center slit that is partially covered with a small rectangular polarizer. The size of the slit/polarizer combination is different for each objective of different magnification; hence the need for a turret arrangement.
HOFFMAN MODULATION CONTRAST i - HMC
• The modulator has three zones that are depicted in Figure 2: a small, dark zone near the periphery of the back focal plane which transmits only one percent of light (areas marked "D" in Figure 2); a narrow gray zone which transmits 15 percent (areas marked "G" in Figure 2); and the remaining clear or transparent zone, covering most of the territory at the back of the objective, which transmits 100 percent of the light (areas marked "B“)
Objective
Condenser
Three-dimensional appearance dictated by phase gradients
DIC / Nomarski • An excellent mechanism for rendering contrast in transparent specimens,
differential interference contrast (DIC) microscopy is a beam-shearing interference system in which the reference beam is sheared by a minuscule amount, generally somewhat less than the diameter of an Airy disk.
• The technique produces a monochromatic shadow-cast image that effectively displays the gradient of optical paths for both high and low spatial frequencies present in the specimen. Those regions of the specimen where the optical paths increase along a reference direction appear brighter (or darker), while regions where the path differences decrease appear in reverse contrast.
• As the gradient of optical path difference grows steeper, image contrast is dramatically increased.
Phase Contrast• Light that is travelling through part of a specimen and is not absorbed by amplitude
objects will not produce a clearly visible image. The intensity remains the same, but the phase is changed compared to the light just travelling in the surrounding areas. This phase shift of about a quarter wavelength for a cultured cell is not visible to our eyes. Therefore, additional optical elements are needed to convert this difference into an intensity shift. These optical elements create a contrast where un-deviated and deviated light are ½ wavelength out of phase, which results in destructive interference. This means that details of the cell appear dark against a lighter background
LIGHT Phase change
Diagram
Principles of DIC / Nomarski
Visualisation TechniquesPhase Contrast
Alignment of Visualisation Techniques- off axis aberration in phase contrast
Objective for Phase Microscopy
Diagram
Principles of DIC / Nomarski
Differential Interference Contrast (DIC)Visualisation Techniques
Alignment of Visualisation Techniques- off axis aberration in phase contrast
HARDWARE MICROSCOPYMicroscopes Stereo and Compound
• Compound Microscopes use two lenses to focus light. 1st lens is the one nearest the object, 2nd lens is the one by the eye
• Total magnification = objective x eye pieces (4x 10x = 40) through the eye pieces
• Limit of resolution for light microscope is 1000x
• 3 different types relevant to our work in IVF
• Binocular - Two microscopes in one – offers observation of specimens at the natural conversion angle of our eyes. This makes the 3D topography visible
Light Microscope Schematic diagram
of a light microscope outfitted for
photomicrography
And what’s the difference between this and the invert ?
Stereo Microscopes
• Variety of Illumination options
• Transmitted Brightfield, Darkfield, Oblique and Polarised Light
• Fluorescence
• Reflected through the optics or by separate source
• Fibre optic goose neck or ring lights
Stereo zooms in IVFTypically either mounted in a class II Cabinet with the cabinet companies light source or as a stand alone unit on the microscopes light base. Uses Brightfield
as standard.
Stereo Microscopes• 2 separate optical paths at an angle to each other
• Brain merges the 2 images to give a 3D image .
• Magnification range 4x- 400x
• 2 principal types
• Large working distances required
Greenhough Optics
• 10 Degree Converging light path
• Great depth of focus
To see objects within a microscope in a similarway, both eyes have to each be provided with a separate light path to enable observation of the specimen at an angle similar to the one our eyes see from naturally
Galilean Optics
• Parallel Optics• Allows build up
of other accessories
Inverted Microscopes
• Predominately used for looking at specimens in suspension- Live cells – ICSI
• Requires Long working distance optics for manipulators
• Whole variety of contrast techniques available
• Environmental and temperature control on stages
• Sample Manipulation/Injection
• Magnifications 40x – 1000x (7000x with additions)
• Typically only found with micro manipulators in IVF lab
• Featured here with the Integra base from RI
• Large body of evidence supporting the importance of anti vibration platforms combined with all manipulators
• Perfect set up and maintenance easy to achieve for both the Integra and the Microscope with the right training
Inverted Scopes in IVF
Upright Microscopes
• Samples on glass slides (Andrology)
• Small Working distance
• Variety of Contrast techniques used
• Magnification range 10x – 1000x
The Microscope- What is Important?
• Location – Ideally in an area with few disturbances
• Avoid direct lighting - not next to a window
• Ensure access to working parts
• Comfortable working area for using both eyepieces and manipulators
• Adjustable chair for different eye heights
• Isolate external vibration – important for manipulation and laser techniques
• Consider an ergonomic head for the microscope
Imaging in the IVF Laboratory
• The image from an optical microscope can be captured by normal light-sensitive cameras to generate a micrograph
• Additional magnification can be achieved using imaging
• Originally images were captured by photographic film but modern developments in CMOS and charge-coupled device (CCD) cameras allow the capture of digital images
• Purely Digital microscopes are now available which just use a CCD camera to examine a sample, and the image is shown directly on a computer screen without the need for eyepieces
Beam Splitter / Prism
CCD Camera• Charged Couple Device - CCD• Cameras are have microscope specific mountings
on them which often feature a demag (x0.55/x0.7) to ensure the image is identical on the screen to what the scope sees
• Current trend is to mount the system on a PC for image manipulations or addition to a PIMS
Camera CCD
C-Mount
Mounting (Demagnification)
Cleaning and Care• Never touch the lenses with your fingers. Your body produces an oil that smudges the glass
• Oil can even etch the glass if left on too long
• Never use paper or tissue to clean the glass of the microscope as they WILL scratch them
• Use only approved Lens or cleaning tools to clean the glass*
• *Avoid Alcohol on LCD displays as this may cause cracking*
Break
Manipulators
START OF OUR PRACTICAL ICSI SESSIONS
Please find yourself a system.
ControlsCourse Controlx and y control10 micron resolution4mm x and y travel
Fine ControlXYZ movement
Sub-micron resolution5mm z travel
XYM
Stage
PL30 and MPH &Tool Holders
PL30
MPHMicro Pipette Holder
Toolholderangle adjustment- 15 to 40 degrees. Vertical, axial and rotational movements
Tool holders
• The tool holders provide the simplest and quickest method ever for setting-up micropipettes. The tool holders, when used with a special objective and spacer (supplied), allow the micropipettes to be set-up 14mm above the Petri dish and then rapidly lowered to the desired position, preventing micropipette damage.
• The tool holders also have a special angular adjustment which allows the user to adjust the angle of the pipette with a single control without moving the tip of the pipette. Unique to RI, this feature makes it easy to ensure that the micropipette is slightly “toe-down” during sperm immobilisation and horizontal during sperm injection
Pipette Challenge
Time to practice with the rigs
Spend time with the opticalpath arrangements of each scope
Understand what causes the optical changes you are seeing
By the end of the day we will all have atime on the board for pipette set up
BEST TIME 3.10 sec / Montana Diaz Diaz – Kings College London
What we are looking at is not always what it is and sometimes it is very well hidden!
The art of good IVF microscopy is effective differentiation
Day 1 MorningWednesday• DAY 1: 8.00 – 9.30 Reception
The office will be open and able to receive trainees for Tea /Coffee reception (for early arrivals!) til 9.30am Please arrive each day in advance of the scheduled start time so we can start promptly
Some attendees will have arranged for RI to pick them up from local accommodation - This will be carried out at this timeLocation: RI Head Office
• 9.30 – 10.15 – Directors and UK Managers welcomeIntroductions and reception with a tour of the facilities and transfer down to the training Suite at Site BLocation: Boardroom RI Head Office
• 10.15 – 10.30 – Introduction and Training aims for the practical sessionTraining aims and individual itinerary discussionFamiliarisation with the format and availability of the range of equipmentLocation: Training Suite Site B. The rest of the sessions will take place here
• 10.30 - 11.30 Session 1 Microscopy – Basics (Nikon, Olympus, Leica)Fundamentals of microscopy work and sample illumination
• 11.30 – 11.45 Short Break• 11.45 – 12.15 Session 2 Microscopy – IVF
Principles for Microscopy in the IVF application including proper set up of Condenser Illumination, Microscope and Optics
• 12.15 – 12.45 Fundamentals of Micromanipulation Integra and Manipulation techniques
• 12.45 – 13.45 Lunch – Off Site Location
Day 1 Afternoon• 13.45 – 15.45 Micromanipulation Continued
– Practical session on the use of micromanipulators - including but not exclusively:• Pipette selection and set-up• Quick set up guide of Pipettes on stage• Understanding the TDU• Understanding the PL30 and MPH• Overview of the HUD and Heated stages – Limitations and Benefits of ITO surfaces• Trouble shooting for common errors in set up in an IVF Lab
• 15.45 – 16.00 Short Break• 16.00 – 16.45 System and Pipette set up challenge
A fun recap on the practical learning's of the day’s study
• 16.45 – 17.00 Close of Day Key learning's and Transfers to accommodation
• 19.30 - Late Evening Events – Smart CasualDinner and Drinks in a seasonally appropriate venueDetails to be confirmed in the morning with the Students
END OF DAY 1
• Transfer to Hotels with Rob & Martyn• Confirmation of Hotel• Pick up to be agreed with each of you.• Please make sure you have our mobile numbers
• Food and Drink provided by RI• 19.30 - Late Evening Events – Smart Casual
Tomorrow Morning
• 8.30- 8.45 am PICK UP at Hotels for those who need it
– Train transfers in the afternoon will be made direct from RI so please bring your cases etc
– If you need to leave early for any reason please let us know and we will try to compress your individual training needs
–Session starts at 9AM!
Day 2
Day 2 Morning
• Thursday• Day 2 • 8.30 – 9.00 Hotel Transfers to RI Training Suite• 9.00 – 10.00 Meeting Start - Manipulator Mania
A fun, practical challenge for two teams to enhance and develop both the understanding and use of the main assemblies and tools in micromanipulation learnt on day 1 and applied in these day’s sessions
• 10.00 – 11.00 Introduction to Advanced TechniquesBiopsy and IMSI use in IVF with the RI Saturn ACTIVE and RI IMSI
• 11.00 – 11.15 Short Break• 11.15 – 12.45 Manipulation and Biopsy I
Instruction on using laser assisted techniques and softwareOpen session and material available for a wide range of techniques either building on the previous days learning's or the start of new skills on more advances equipment
• 12.45 – 13.15 Lunch – On/Off site catering
I NEED A FAVOUR...
Day 2 Afternoon• 13.15 – 13.45 Manipulation and Biopsy II
Further hands on practical time and practice on the latest available ICSI/PGD and IMSI equipmentThroughout the sessions and during time when other equipment is taken up with other students instruction for use of the following environmental monitors will be available
• 13.45 – 1400 Short Break and Sugar• Cornwall’s best tradition to keep us going
• 14.00 – 15.15 Individual Development Session – The Final Session!Individual itinerary focus for students to cover any aspect of the two days as they and the trainer see fitThere will be almost 1-1 coverage of each aspect of the two days learning at each work areas with a wealth of RI trainers available for support
• 15.15 – 15.30 Close of Workshop and ThanksTransfer to accommodation or trains and departures
• 15.30 – Late After HoursThe RI UK team will be available in the training suite for a further hour for those staying onAnyone staying another night in Falmouth are welcome to join us again for an evening out
Integra Ti components
Syringes
Temperature variation with objectives
34.0
35.0
36.0
37.0
38.0
39.0
40.0
ITO Heated Stage with Objectives (4x 10x 20x) Bourn Hall 13/04/12
20x rotated10x rotated
4 x in position
Ref 1
Parfocal Alignment
Alignment of Olympus Relief or Hoffman Modulation
Biopsy Session -
IMSI Session
Microscope Workshop
In Association with Research InstrumentsNikon, & Olympus Microscopes
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