Digital Imaging in Microscopy

Hand DrawingThe Camera Lucida

Film at focal plane of projection lens

Photo tube

Projection Lens

• 4x5" film• 35 mm film

• Black & White filmTechPan 100

• Color filmK25, K64

Film Photography

First PassSecond Pass

∆T = 1/60 s (fields)

Full frame image = 1/30s

Video Imaging — Analog TechnologyDynamic visualization

SignalCurrent

Video SignalVoltage

Target VoltageSupply

10-100v

Target Electrode Photoconductive Layer

Electron beam

Cathode

Filament 525 l

ines

Interlacing

TV Standards

International Television Standards Committee1950s

NTSC, PAL, SECAM

• 525 TV lines• 60 Hz field rate

Realized resolution = 350 horizontal lines

Digital Imaging using Analog Cameras

rateanalog-to-digital

NTSC analog video

Frame grabber

Resultant digital image is 640x480 pixels

Imaging Requirementsfor

Fluorescence Microscopy

Specimen

Excitation filter

Dichroic mirror

A B

Objective

Barrier filter

Fluorescentlight fromsample

Arc light source

Epifluorescence microscope with color image capture

Low-light Image Capture

Increasing Light Sensitivity of Video Detectors

Silicon Intensified Target (SIT) Camera

Vidicon image intensifier

NTSC video<350 lines resolution

Charge Coupled Device (CCD) Detectors

Front-illuminated CCD

Solid State image detectors

Color Imaging — 1 CCD

Analog (0.6 MPix) 1995

1/3” Sony frame transfer CCD

Bayer color filter

Color Imaging — 3 CCDs

1

2

3

CCDsFiltersDichroics

Analog (640x480) Technology1.2MPix

CCD size

Bit Depth and Dynamic Range

8-bit

12-bit

Greater Bit-Depth and Dynamic Range Records More Information

Quantum Efficiency and Spectral Response

QE vs. Impinging wavelength

QE = photons/electrons

Backside-illuminated CCD

Increasing Quantum Efficiency (QE) of CCD

Etching uniformly thins a CCD to a thickness of approximately 10 µm. The image is focused on the backside of the CCD register where there is no gate structure. Thinned CCDs exhibit high sensitivity to light from the soft x-ray to the near-infrared regions of the spectrum.

Coating the CCD can improve the spectral sensitivity of the CCD (top: coated)

Increasing Spectral Response of CCD

Increasing Signal Readout Rate Interline Transfer CCD

Serial Clocks

ParallelClocks

Output Amp

direction of parallel shift

Dark Current and CCD Temperature

Single-Stage Peltier Cooling-10°C ambient

Optronics DEI 450

Photometrics CH350 digital camera

Two-Stage Cooling

Heat from Peltier drawn off by recirculating liquid

Two-Stage Peltier Cooling-35°C to -50°C

Resolution and Sampling

amplitude quantization

N<N>N

Sampling rate (1/T)

Nyquist sampling

1/T > 2f

The rate of sampling must be at least twice the frequency of the resolvable desired structure (signal)

300 dpi 72 dpi 20 dpi

Matching microscope resolution and CCD pixel size

1. Determine microscope resolution d=0.61λ/NA

where NA is the average of the objective and condenser

2. Match the microscope resolution with (a minimum) of two pixels on the CCDM• 0.61λ/NA = 2.0p

where M=the objective magnification; p=pixel sizeor

M = 2p/(0.61λ/NA)

e.g., CCD with 6.8µm pixels and green light (500nm) illumination:M = 2•6.8μ/(0.61•0.5μ/1.3)

M=58Thus, a 60x, 1.3 N.A. microscope objective provides a diffraction-limited image

for the many CCDs (e.g., KAF1401E CCD, QImaging).

Photometrics QuantixKAF 1401E

1035x1317 pixels6.8µm x 6.8µm pixels 12 bit pixel depth5MHz readoutTwo-Stage cooling to -50C

Frame readout CCD

Photometrics CoolSnap HQfor Fast Imaging

1040x1392 pixels12-bit pixel depth6.45µm x 6.45µm pixels 20MHz readoutSingle-stage cooling to -30C

Interline transfer CCD

Sony ICX282 Progressive Scan Interline CCD, Color2560 x 19203.4µm x 3.4µm10-bit20, 10, 5, 2.5MHzPeltier thermoelectric cooling to -10°C below ambient

QImaging MicroPublisher

5 MPixBayer filter CCD

Digital Images

(0,0)

(1024,1024) Blue

Red

Green

E. herbico -la

299R (P fruB -gfp

[AAV])

-3

-2

-1

0

1

2

3

0 2 4 6 8 10 12

cube root [mean pixel intensity]

t=0 (inoculum)

t=4 (leaf #2)

t=25 (leaf #1)t=50 (leaf #1)t=75 (leaf #1)

50

25

105

10.5

75

9095

9999.5

Digital Image Processing and Analysis

Digital Segmentation

and Image Analysis

Some Applications Require 12-16 Bit Dynamic Range

Sarah Swanson, Jones lab PMB

ABA

GA

DIC 440nm fluor ratio

Confocal MicroscopesPoint-Source Detectors (PMT)

Ch 1 (32 PMTs)

Ch 2

Ch 3

pinholesDichroics

Sample Plane

Laser

TransmittedLight detector

diffraction grating

C o n f o c a l M i c r o s c o p y G e n e r a t e s Vo l u m e t r i c D a t a

Software Generates 3D Projections through Volumetric Data

Green: DAPI-stained ChromosomesRed: 5S rDNA

5S sequence marks the end of chromosome 5

3D Projection Using Voxel Data

Ring canals between nurse cells and oocytes

Red:Rhodamine phalloidinBlue: DAPIGreen: FITC-Ab

V o l u m e t r i c D a t a C o l l e c t i o n A l l o w s f o r 3 D I n v e s t i g a t i o n

leech segmented central nervous

system

The nuclei of all the progeny,

neurons and glial cells, are labeled

GFP Expression in Arabidopsis roots

Arabidopsis mutant from J. Haseloff Lab

Feldman lab, PMB

Deconvolution Microscopy

PSF

3D Image Processing and Analysis

Yeast Golgi

Pixel Colocalization

2D histogram

Binary channel

T h e G o l g i A p p a r a t u s

Localization of Golgi and trafficking molecules

Sebastien Carreno, Drubin Lab

Giardia as Imaged using Deconvolution Microscopy

Isosurface modeling

Deconvolved voxel projection

Red: nucleiGreen: fluorescein anti-tubulin