Optical and Biological Considerations for Confocal Microscopy

Steven Ruzin Ph.D.CNR Biological Imaging FacilityUniversity of California, Berkeley

Specimen

Excitation filter

Dichroic mirror

A B

Objective

Barrier filter

Arc light source

E p i f l u o r e s c e n c e M i c r o s c o p e

Diffraction and Out-Of-Focus light

d

d = Airy disk (1 Airy unit)

Out-of-focus information

Marvin Minsky’s 1957 Confocal Microscope

Specimen d

Pinhole diameter = d; excludes diffraction rings

Source pinhole

Pinhole Diameter and Confocal slice thickness

Sarastro 1989 Confocal Microscope Design

d

xy

Objective

Scanner

Laser

Lens

Object plane

Detector 2

Dichroic Beam Splitter 1

Focus motor/ “Stepper Motor”

Dichroic Beam Splitter 2

Detector 1Pinhole

d

Ch 1 (32 PMTs)

Ch 2

Ch 3

pinholesDichroics

Sample Plane

Laser

TransmittedLight detector

diffraction grating

Zeiss LSM Meta; Leica SP2, Olympus FV1000

Contemporary Laser scanning Confocal Microscope Design

Fluorescence Emission Properties

DAPI SYTO 11

Stokes Shift

Discriminating Fluorescent Probes

A-488 A-594 A-488 A-546 A-568 A-594

Molecular Probes’ Alexa Dyes

Fundamental Limits to Confocal Microscopy

Registration of multiple probes Fluorescence limitations

Sample Prep

Compensating for Varying Section Thickness at Multiple Wavelengths

Adjusting the Optical Slice Thickness

Barley Aleurone protoplast

Adjust pinholes to normalize optical slice thickness

Pinhole Adjustment yields:• Sections in-register

• Normalized confocal slice thickness

C. elegans Z. mays

The problem of simultaneous UV/Vis excitation

UVVis

Collimator adjustment for separate laser fibers

Transcriptional foci in Drosophila imaged using UV & Vis excitation

Nuclei (DAPI/ UV)

Transcriptional foci of 2 linked

genes (488/543nm)

Separate UV & Visible light paths can image different focal planes

UV/Vis Ex misalignment

UV-excited and Vis-excited probes imaged to the same plane

BacteriaDAPI and Autofluorescence

A Few Other Potential Problems

Standard coverslip designations

#1 = 130–170µm#1.5 = 160–190µm#2 = 170–250µm

Variation in Coverslip Thickness

Objectives are most efficient when the correct coverslip is used

Barley

Mismatched coverslip thickness results in decreased energy, reduced depth resolution, and axial shift in focus

Autofluorescence

Z. mays

Insect leg

Autofluorescence

Ex 488Em 505–550

Ex543Em LP560

??Paraffin-embedded

Ex 488, 543Em LP560

Aldehyde-inducedLung, H&E

Photobleaching

Single scan Multiple scans (L)

Antifade Solutions

• Commercial: “ProLong”, “SlowFade”• DABCO 5–15mg/ml• p-phenylenediamine 1mg/ml• n-propyl gallate 10–50mg/ml

Traditional Fluorescent Probes

HOOC CH2CH2

HOOC

COOH

OHO

CH2CH2 COOH

O

BCECF

Mg2

C

OCH3OC20H39

CH3

I II

IV III

VHH

OOC O

H

NN

Chlorophyll

NH2

H2NC

C

NH2

NH2NH

DAPI

OCH3C

O O

O

O

CO CH3O

FDA

H2N

NH2

H3C

CH3CH3

N

I-

N

PI

+ +

I-

H2N NH2

COOCH3

Rhodamine 123

CH3OOC CH2OOC CH2CH2

HOOC

COOH

OHO

CH2CH2 COOCH23COOCH3

O

BCECF-AM

Contemporary Fluorescent Probes: Alexa Dyes

Alexa 488O

C

NH2H2N O

SO3SO3

OH

O

O

- -

+

-

Environmental Sensitivity: pH

Sample Preparation

• Mountant contains glycerol and antifade solution• Seal coverslip edges well• Remove “old” immersion oil before re-viewing• Refreezing is not recommended

Old Oil Absorbs Water

n = 1.443n = 1.438

Immersion oil = 1.479–1.52

Slide stored at -20C ≥ 1 month

Sampling Techniques

Nyquist Sampling

128x128, 4x Average 512x512, 4x Average

Rate ≥ 2f(highest)

Averaging pixel intensity can increase signal-to-noise

Pixel Averaging

The Color Lookup Table“Let me just take a look at it.”

Imaging System Assigns Colors Based on Assumptions

Thank you

Giardia