Confocal Microscopy

Chaitanya namdeo

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

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

Epi-illumination

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

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

Tissue histology Blood cells

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)

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 grain fibers

Principle of confocal microscopy

In 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

OUT-OF-FOCUS PLANE IN-FOCUS (OBJECT) PLANE CONTAINING ILLUMI NATED SPOT OUT-OF-FOCUS PLANE

"POINT" SOURCE OF LI GHT CONDENSER LENS BIOLOGICAL SAMPLE OBJECTIVE LENS

"POINT" DETECTOR APERTURE

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 galvanometricdriven mirrors

Optical train of a confocal microscope

LASER BEAM

BEAM SPLITTER

TARGET SURFACE

RASTER PLANE

GALVANOMETRIC SCANNER

RASTER LINE

MICROSCOPE OBJECTIVE

POLYGON SCANNER

CONFOCAL SCANNING LASER MICROSCOPE

Optical train of a confocal microscope

AVALANCHE PHOTODI ODE WI TH PI NHOLE

LASER BEAM

BEAM SPLITTER

TARGET SURFACE

RASTER PLANE

GALVANOMETRIC SCANNER

RASTER LINE

MICROS COPE OBJECTIVE

POLYGON SCANNER

CONFOCAL SCANNING LASER MICROSCOPE

LASER BEAM VIDEOTAPE RECORDER AVALANCHE PHOTODI ODE WI TH PI NHOLE

BEAM SPLITTER

VIDEO MONITOR

FRAME GRABBER TARGET SURFACE RASTER PLANE RASTER LINE

GALVANOMETRIC SCANNER

MICROSCOPE OBJECTIVE

POLYGON SCANNER

CONFOCAL SCANNING LASER MICROSCOPE

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

http://www.olympusfluoview.com/java/scanningmodes/index.html

Pollen grain

Hamster ovary Mouse intestine cells

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 antibody Study 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, cellenvironment 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

ROTATABLE HEAD MECHANICAL ARM 3-AXIS TRANSLATION STAGE OBJECTIVE LENS HOUSING RING-AND-TEMPLATE (attached to skin and locks into the housing)

VivaScope by Lucid

Courtesy of S. Gonzalez

Reflectance-mode Confocal Microscopy

Live Normal Skin

En face SECTION
H&E Confocal in vivo

SC SG

SS

DEJ

VERTICAL Hematoxylin & Eosin stained section of tissue

Rajadhyaksha M, Gonzlez S, et al. J Invest Dermatol 1999;113;293-303. 180 m 100x, 1.2NA

Courtesy of S. Gonzalez

Elongated nuclei Monomorphism Uniform Polarization of nuclei

60 x, 0.85 NA x

20 m

Stained in vitro section

In vivo confocal

250 m

Courtesy of S. Gonzalez

OVERALL SENSITIVITY AND SPECIFICITY

Criteria
Elongated monomorphic nuclei

Sensitivity %
100 92 83 88

Specificity %
71 97 55 54

Polarized nuclei Inflammatory infiltrate Increased vascularity

Pleomorphism
2 or more criteria 3 or more criteria

64
100 94

64
54 78

4 or more criteria

83

96

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