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Super-Resolution Microscopy: Test Samples

Dan Metcalf

Biotechnology Group

National Physical Laboratory

PDF compression, OCR, web optimization using a watermarked evaluation copy of CVISION PDFCompressor

http://www.cvisiontech.com/

Outline

1. NPL > Biotechnology > Super-resolution

2. Localisation microscope hardware (dSTORM)

3. Sample preparation (dSTORM)



History



Acoustics

Advanced Materials

Analytical Science

Biotechnology

Electromagnetics

Engineering Measurements

Environmental Measurement

Ionising Radiation

Mathematics & Scientific Computing

Nanoscience

Optical Radiation & Photonics

Quantum Phenomena

Time & Frequency



Biotechnology Group



Super-Resolution Localisation Microscope

Blinking or fluctuating signal

• dSTORM - dyes

• PALM - FPs (PA & PS)

• SOFI - dyes, FPs & QDots

• BaLM - any fluorescence*

*needs to bleach or blink

Structured Illumination Microscope

Continuous fluorescence

• Flexible fast multi-colour 3D design

• Live cell imaging



Super-Resolution People

Alex Knight Super-resolution PRS, software

Miklos Erdelyi Optics

Dan Metcalf Cell biology, sample preparation, user

Kevin O’Holleran Optics, software

Mike Shaw Optics, software

Neelam Kumarswami Biochemistry, sample preparation, user

Rebecca Edwards Biology, sample preparation, user

Clemens Kaminski Laser Analytics PI

Miklos Erdelyi Optics

Eric Rees Software, chemistry

Simon Jobst Software

Gabi Schierle-Kaminski Biology, sample preparation



Localisation Microscope

Lasers & optics 405 nm (100 mW)

488 nm (150 mW)

642 nm (150 nm)

Super-continuum (450 nm – 650 nm)

TIRF mirror TIRF, HILO, Epi illumination

EMCCD Inverted microscope Covered motorised piezo-Z stage

Foil

Coming soon 3D astigmatism Focus lock



Test Sample Preparation Choose a test sample

• Epidermal growth factor (EGF) in HeLa cells

• Actin – in vivo & in vitro

• Dextran – in vitro

Choose a dye(s)

• Alexa 647, Cy5

• Atto 488, Alexa 488, Atto 520, Abberior CAGE 500

Choose a labelling method • Immunolabelling - Fab, Fab(2), antibody

• Direct conjugate – phalloidin, EGF etc

Choose a format • LabTek chamber

• Coverslips PDF compression, OCR, web optimization using a watermarked evaluation copy of CVISION PDFCompressor


Choose a format…

LabTek chamber Easy buffer exchange

Coverslip & cavity slide Coverslip flexibility eg. correlative EM

Coverslip & ring chamber Small coverslips But grease = movement



Choose a labelling method…

Antibody F(ab’)2 Fab Small protein

eg. EGF/phalloidin

Direct dye conjugation eg.

actin-A647

~20 nm = 1

& 2

antibodies

~ 1 nm = direct dye conjugation



Choose a dye…

Different dyes: Different chemistries Different buffers/mounting conditions Different acquisition parameters PDF compression, OCR, web optimization using a watermarked evaluation copy of CVISION PDFCompressor


Localisation precision Alexa 647 Atto 655 Cy 5.5



Dye issues

• Thiol, concentration, oxygen scavenger?

– Contradictory protocols published

• Degree of labelling issue

– Zhuang, Sauer, Hell custom labelled DOL = 1

– Commerical = 3-8

• Current sample analysis work

– In vitro samples

– Software metrics (data quality)

– Commercial & custom-made dye conjugates (Alexa 488, Atto 488, Atto 520)



Test sample: EGF in HeLa cells

Seed HeLa cells

1. Fix in 4% formaldehyde for 7 min 2. PBS wash x 2 3. Stain with EGF-Alexa 647 4. PBS wash x 2

Staining protocol

EGF labelling Cell surface receptors clathrin coated pits 1. Fill with buffer & seal

2. Find a cell of interest 3. Reference image(s) 4. Acquire dSTORM raw data 5. Process to reconstruct super-

resolution image

dSTORM imaging



dSTORM raw data

160 nm pixels

64 p

ixels

= 1

0.2

4 μ

m



TIRF - 512

TIRF - 64

Sum

dSTORM



Clathrin coated pits (50-100 nm)

Individual ‘EGF vesicles’ (50-100 nm)



Test sample: Actin in vivo

Phalloidin-Alexa 647 HeLa cells

HILO -512 HILO - 64



Discontinuous filaments



Phalloidin-Alexa 647 labelling in vitro actin filaments

Test sample: Actin in vitro



0-10000 frames

1-1000 frames

9001-10000 frames

Drift Analysis



Standards & References

Dye testing Staining optimisation eg. immunolabelling protocols Microscope performance Localisation software performance Acquisition settings Data quality

Microscope comparisons (hardware & software) Microscope maintenance Training & controls



[email protected]

[email protected]

www.npl.co.uk/biotechnology



super-resolution microscopy: test samples · pdf filesuper-resolution microscopy: test samples...

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