Lecture 19

Principles of optical microscopy

Illumination conjugate planes are shown in red; an image of the lamp filament is in focus at these planes

Imaging path conjugate planes are shown in red; an image of the specimen is in focus at these planes

http://www.microscopyu.com/articles/formulas/formulasconjugate.html

http://www.microscopyu.com/tutorials/java/conjugateplanes/index.html

Transillumination

Field diaphragm: will affect size of field that is illuminated at the focal plane

Condenser aperture: will affect the numerical aperture of the condenser

Field diaphragm: will affect size of field that is illuminated at the focal plane

Aperture diaphragm: will affect the numerical aperture of the objective for illumination

Epi-illumination

Objective specifications

Brightfield or trans-illumination microscopy

• Simplest type of microscopy

• Contrast provided by absorption

• Biological specimens are not highly absorbing naturally

• Use stains, which typically require fixation, i.e. cells no longer alive

• Used routinely in histopathology and hematology and basic science studies for which looking at live specimen is not crucial

Blood cellsTissue histology

Phase contrast microscopy• First microscopic method which allowed visualization of

live cells in action• Nobel prize in physics was awarded to Frits Zernike in

1953 for its discovery• It enhances contrast in transparent and colorless objects

by influencing the optical path of light• It uses the fact that light passing through the specimen

travels slower than the undisturbed light beam, i.e. its phase is shifted

Phase contrast microscopy• Let S (red) be light passing through

medium surrounding sample and D (blue) light interacting with specimen. S and D typically interfere to yield P (green), which is what we can usually detect.

• P will be phase shifted compared to S, but our eyes cannot detect phase shifts.

• Phase contrast microscopy effectively converts this phase shift into an intensity difference we can detect

Phase contrast microscopy

• Condenser annulus allows only ring of light to reach the condenser.• Light rays illuminating the sample but not interacting with it will go

straight through and be imaged along a ring at the back focal plane of the objective

• Light rays diffracted by the sample are phase shifted by a approximately a quarter wavelength and will be scattered over a range of angles and will generally not propagate in the exact forward direction

• A phase plate at the back focal plane of the objective alters selectively the phase and magnitude of the non-diffracted wave.

http://www.microscopyu.com/articles/phasecontrast/phasemicroscopy.htm (reference)l

Phaseplate

http://www.microscopyu.com/tutorials/java/phasecontrast/positivenegative/index.html (ex)

Differential interference contrast• Differential Interference Contrast

(DIC) microscopy converts phase shift gradients across different parts of a specimen into intensity differences.

• Doesn’t suffer from some artifacts seen in phase contrast

• Uses full NA of objective

http://www.olympusmicro.com/primer/techniques/dic/dicintro.html

• Mouse fibroblast embryo, 24.3 hour time lapse video

http://www.microscopyu.com/moviegallery/livecellimaging/3t3/index.html

DIC image of nematode embryo

Principle of fluorescence

Principle of Fluorescence1. Energy is absorbed by the atom which becomes excited.2. The electron jumps to a higher energy level.3. Soon, the electron drops back to the ground state, emitting a photon (or a packet of light) - the atom is fluorescing.

Fluorescence Stoke’s shift

• Fluorescence emission peak wavelength is red-shifted with respect to absorption peak wavelength

• This shift may vary typically from 5 to more than 100 nm, depending on the electronic structure of the molecule

Advantages of fluorescence• Highly sensitive method• Simple implementation• Highly sophisticated fluorescent probes

– Fluorescent dyes that accumulate in different cellular compartments or are sensitive to pH, ion gradients

– Fluorescently tagged antibodies to specific cell features– Endogenously expressed fluorescent proteins

» Really endogenous NADH/FAD: enzymes involved in ATP

productionstructural proteins: collagen/elastinamino-acids: tryptophan/tyrosine

» After gene modificationGreen fluorescent protein and variants

Optical path of fluorescence microscope

http://www.microscopyu.com/articles/fluorescence/fluorescenceintro.html

Dichroic filter: reflects excitation and transmits fluorescence

You can image simultaneously or sequentially the same sample at different excitation emission wavelengths to look at

different cell components • Cell nucleus stained

with blue Hoechst dye

• Mitochondria stained with Mitotracker red

• Actin cytoskeleton stained with phalloidin derivative conjugated to Alexa 488 (green)

Photobleaching often limits the number of exposures or the exposure time

Photobleaching is the irreversible photochemical destruction of the fluorescent chromophores

Resolution is limited in thick specimens by detection of out-of-focus fluorescence

• In a standard fluorescence microscope, the excitation beam illuminates uniformly a wide field of the sample.

• If the sample is thick, fluorescence will be excited within the focal plane, but also within planes above and below the focus.

• Some of this fluorescence will be imaged onto the detector and will result in a defocused-looking image

Human medulla rabbit muscle pollen grainfibers

Principle of confocal microscopyIn confocal microscopy two pinholes are typically used:

– A pinhole is placed in front of the illumination source to allow transmission only through a small area

– This illumination pinhole is imaged onto the focal plane of the specimen, i.e. only a point of the specimen is illuminated at one time

– Fluorescence excited in this manner at the focal plane is imaged onto a confocal pinhole placed right in front of the detector

– Only fluorescence excited within the focal plane of the specimen will go through the detector pinhole

– Need to scan point onto the sample

CONDENSER LENS

OBJECTIVE LENSBIOLOGICAL

SAMPLE

OUT-OF-FOCUS PLANE

OUT-OF-FOCUS PLANE

"POINT" SOURCE OF LIGHT "POINT"

DETECTOR APERTURE

IN-FOCUS (OBJECT) PLANE CONTAINING ILLUMINATED SPOT

To create confocal image, scanning is required

• Either specimen is scanned past excitation beam or laser beam is scanned across specimen

• For biological experiments, it is most common to scan the laser beam across focal plane using a combination of two galvanometric-driven mirrors

POLYGON SCANNER

GALVANOMETRIC SCANNER

MICROSCOPE OBJECTIVE

RASTER LINERASTER

PLANETARGET SURFACE

LASER BEAM

BEAM SPLITTER

CONFOCAL SCANNING LASER MICROSCOPE

Optical train of a confocal microscope

POLYGON SCANNER

GALVANOMETRIC SCANNER

MICROSCOPE OBJECTIVE

RASTER LINERASTER

PLANETARGET SURFACE

LASER BEAM

AVALANCHE PHOTODIODE WITH PINHOLE

BEAM SPLITTER

CONFOCAL SCANNING LASER MICROSCOPE

Optical train of a confocal microscope

POLYGON SCANNER

GALVANOMETRIC SCANNER

MICROSCOPE OBJECTIVE

RASTER LINERASTER

PLANETARGET SURFACE

LASER BEAM

AVALANCHE PHOTODIODE WITH PINHOLE

FRAME GRABBER

VIDEO MONITOR

BEAM SPLITTER

VIDEOTAPE RECORDER

CONFOCAL SCANNING LASER MICROSCOPE

Elimination of out-of focus fluorescence yields superior images

http://www.olympusfluoview.com/theory/confocalintro.html

A thick specimen can be optically scanned in three dimensions and the images can be processed to yield cross-sections along plane of interest, three dimensional composites and animations

Pollen grainHamster ovarycells

Mouse intestinehttp://www.olympusfluoview.com/java/scanningmodes/index.html

In vivo depth-resolved imaging is possible

Tumor cells grown subcutaneously in mice, expressing Green Fluorescent Protein

Blood vessels stained with Cy5-conjugated anti-PECAM antibodyStudy interactions of tumor cells with their environment and potential

factors/drugs that affect processes, such as tumor growth or metastasis

Video rate microscopy captures dynamic interactions

• Monitor cell-cell, cell-environment interactions in natural environment to understand animal and human biology and processes involved in disease development

• Monitor dynamic interactions

In Vivo Reflectance Confocal Microscopy of human skin

3-AXIS TRANSLATIONSTAGE

ROTATABLE HEADMECHANICAL ARM

OBJECTIVE LENS

HOUSING

RING-AND-TEMPLATE(attached to skin and locks into the housing)

VivaScope by Lucid

Courtesy of S. Gonzalez

OPTIMUM RANGE PARAMETERS FOR RCM OF HUMAN SKIN

•Tissue Stability M-T clamping fixture

•Illumination Power up to 40 mW

•Imaging Rate 10-30 frames/sc

•Detector aperture diameter 100-200 µm

•Objective lenses 30 -100X, 0.7-1.2 NA

•Refraction index medium 1.33 (water)

•Wavelengths 400-700 nm (visible) 800-1064 nm (NIR)

Confocal in vivo H&E

VERTICAL Hematoxylin & Eosin stained section of tissue

“En face” SECTION

Rajadhyaksha M, González S, et al.J Invest Dermatol 1999;113;293-303.

180 µm100x, 1.2NA

SC

SG

SS

DEJ

Reflectance-mode Confocal Microscopy

Live Normal Skin

Elongated nuclei - Monomorphism

Uniform Polarization of nuclei

20 µm

60 x, 0.85 NA

250 µm

x

y

Stained in vitro sectionIn vivo confocal

Courtesy of S. Gonzalez

Criteria Sensitivity % Specificity %

Elongated monomorphic nuclei 100 71

Polarized nuclei 92 97

Inflammatory infiltrate 83 55

Increased vascularity 88 54

Pleomorphism 64 64

2 or more criteria 100 54

3 or more criteria 94 78

4 or more criteria 83 96

OVERALL SENSITIVITY AND SPECIFICITY

Results remained reliable across study sites and across Basal Cell Carcinoma subtypes.

Combination of clinical photograph examination and reflectance confocal microscopy evaluation significantly improved non-invasive diagnosis of BCC

Courtesy of S. Gonzalez

http://www.aep.cornell.edu/drbio/MPE/mpe.html

Multiphoton microscopy

•At very high photon densities, it becomes possible for two or more photons to be simultaneously absorbed•Each multiple absorption induces a molecular excitation of a magnitude equivalent to the sum of the absorbed photon energies

Multi-photon fluorescence: Basic principles• Multi-photon excitation is a nonlinear process• Because two photons are required for each excitation, the rate of two-photon

absorption depends on the square of the instantaneous intensity.• Because of the large intensities required, high power lasers providing very short

pulses (~100 fs) are used, so that peak intensity is high, but average power doesn’t damage the specimen.

• We have photon flux densities sufficiently high for multiple photons to arrive “simultaneously” (in 10-15 s) at an excitable molecule (of 10-16 cm2 cross section) only at the focus point of a beam.

• The probability that a given fluorophore at the center of a focused beam absorbs a photon pair during a single pulse is

advantagephoton - two theasknown is

frequency repetition theis F

aperture numerical isNA

power average theis

section-cross absorptionphoton - two theis

*

2

2

p

2212

p

p

P

hc

NAFPn pa

Advantages of multi-photon excitation

• With a single-photon source excitation occurs throughout the beam profile

• With a two-photon source excitation events are limited to the beam focus

• Focal point restriction of excitation automatically provides 3-dimensionally resolved submicron information

• Photodamage is restricted to the focal plane• Not necessarily to refocus the fluorescence through an

aperture– Simpler, more efficient optical detection design– Scattering in thick specimens degrades signal to a smaller

extent• UV absorbing molecules can be excited using practical

visible/NIR wavelength ranges

Second Harmonic Generation (SHG)

• SHG can be thought off as the scattering equivalent of two-photon excited fluorescence

• The emitted photons are at exactly half of the wavelength of the incident radiation (as excitation changes, emitted SHG signal also changes)

• The SHG signal is phase matched to the incident radiation and it is emitted in a highly directional fashion, which depends on the size, shape and refractive index of the scatterers. (fluorescence is incoherent and isotropic)

http://www.aep.cornell.edu/eng10_offsite.cfm?URL=http%3A%2F%2Fwww%2Edrbio%2Ecornell%2Eedu

Instrumentationhttp://www.aep.cornell.edu/eng10_offsite.cfm?URL=http%3A%2F%2Fwww%2Edrbio%2Ecornell%2Eedu

Multi-photon imaging is the method of choice for looking at endogenous

fluorescence in thick biological specimens

Two-photon excited fluorescence (TPEF) is particularly useful in imaging of endogenous weak fluorescence

Second harmonic generation (SHG) yields excellent intrinsic contrast for imaging of asymmetric molecules, such as collagen

http://www.drbio.cornell.edu/Infrastructure/MPM_WWW/MPM_hist/home.htm

4-Pi, two-photon fluorescence microscopy

• Combines localized excitation with coherent fluorescence detection to beat resolution limit

• Resolution achieved 80 nm

Mitochondrial network of a live yeast cell

Gugel et al. Biophys J 2004; 87:4146-4152