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Fluorescence microscopy

Fluorescent microscopy fundamentals & applications

Last author update: 1 March 2022
Last staff update: 17 May 2022

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PubMed Search: Fluorescence microscopy [title] applications pathology

Ruhani Sardana, M.B.B.S.
Anil Parwani, M.D., Ph.D., M.B.A.
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Cite this page: Sardana R, Parwani A. Fluorescent microscopy fundamentals & applications. website. Accessed September 22nd, 2023.
Definition / general
  • Noninvasive form of optical imaging which applies the principle of fluorescence in the diagnostic / investigative study of organic and inorganic components of the specimen
  • Enables multiantigen detection and permits live cell imaging (Arch Pathol Lab Med 2011;135:255)
  • Some structures and organisms naturally exhibit fluorescence (autofluorescence), e.g. mitochondria, lysosomes, amino acids
  • Set up can be simple, such as an epifluorescence microscope or complicated, such as a confocal microscope
Essential features
  • Microscope consists of a light source (xenon arc lamp or mercury vapor lamp, LEDs and lasers), an excitation filter, a dichroic mirror (or dichroic beam splitter) and the emission filter (Cold Spring Harb Protoc 2014;2014:pdb.top071795, Genetics 2019;211:15)
  • Principle is to shine light of a particular wavelength (known as excitation) onto a specimen containing a fluorophore and then visualize emitted light (known as emission) at a longer wavelength
  • Fluorescent in situ hybridization (FISH), a powerful diagnostic tool, can detect both structural (translocation / inversion) and numerical (deletion / gain) genetic aberrations
Fluorescence microscopy background
  • Lehman, in 1914, described the first fluorescent microscope
  • Haitinger, in 1935, stained a histological preparation of tissue / smear with fluorescent dyes for the first time (Genetics 2019;211:15)
  • Hageman, in 1937, stained the acid fast bacilli microorganism using fluorescent dyes
  • Microscope consists of a light source (xenon arc lamp or mercury vapor lamp, LEDs and lasers), an excitation filter, a dichroic mirror (or dichroic beam splitter) and the emission filter (Cold Spring Harb Protoc 2014;2014:pdb.top071795, Genetics 2019;211:15)
  • Principle is to shine light of a particular wavelength (known as excitation) onto a specimen containing a fluorophore and then visualize emitted light (known as emission) at a longer wavelength
  • Fluorophores can be biological (green fluorescent protein or GFP), organic dyes (rhodamine, fluorescein, cyanine, etc.) or quantum dots
Fluorescence microscopy design
Fundamentals of fluorescent microscopy
  • Light (or photon) hits fluorophore, which excites electrons
  • The excited electron (high energy, short wavelength) loses energy which translates into emission of light (low energy, long wavelength)
  • Strokes shift: difference between the excitation and emission maxima
Components of fluorescent microscopy
  • Fluorescent dyes: chemical compounds ( DAPI, Hoechst, Phalloidin) which bind to a biological molecule of interest and re-emit light upon excitation (Genetics 2019;211:15)
  • Light source: lasers for complex microscopy; xenon lamps, mercury lamps and LEDs with dichroic excitation filters for epifluorescence microscopes
  • Excitation filter: a bandpass filter which allows only the wavelengths absorbed by the fluorophore, minimizing excitation of other sources of fluorescence
  • Dichroic mirror: highly specific color filter allowing only a small range of colors while reflecting other colors
  • Emission filter: a bandpass filter allows only wavelengths emitted by fluorophore and blocks all other undesired light, especially excitation light, ensuring the darkest background
Types of fluorescence microscopy
  • Epifluorescence microscopy: transmitted and emitted light travel through the same objective lens
  • Confocal microscopy: incorporates optical sectioning, specialized for thick 3D samples
    • Point Scanning: focuses excitation light to a point in the sample
    • Parallelized (spinning disk): more than one hole illuminated at a time, hence faster image formation
  • Total internal reflection fluorescence (TIRF): to excite a very thin layer of fluorescent molecules, lying just under the coverslip
  • Light sheet microscopy: creates a thin sheet of excitation light by using two or more objectives, reduces photo bleaching and helps in 3D imaging (Cardiovasc Res 2021;117:520)
  • Super resolution microscopy: to image below the resolution (diffraction) limit of a light microscope (Nat Cell Biol 2019;21:72)
Diagrams / tables

Images hosted on other servers:

Image generation

Visualization of pinhole principle

Diagnostic applications in microbiology / infectious pathology
  • Rapid diagnosis of different fungi, such as yeast, Microsporum, Trichophyton, etc., especially in immune compromised patients
  • When using culture optical brighteners (ANS) as agar medium additives, the gram negative bacteria are more fluorogenic than gram positive bacteria
Diagnostic application in immunopathology
  • Determining the location of an antigen or antibody in a tissue section or smear by the pattern of fluorescence, using specific antibody or antigen labeled with a fluorochrome (immunofluorescence)
  • Diagnostic and prognostic tool in autoimmune and vesiculobullous lesions of the skin and mucosa (J Oral Maxillofac Pathol 2017;21:402)
  • Particulate antigens, such as bacteria or protozoa, soluble toxins (staphylococcal enterotoxin) and viruses can be demonstrated in tissues or specimens (Front Immunol 2018 Mar 21;9:598)
  • Identify and titrate host antibodies to infective agents and autoantibodies against self through indirect fluorescent antibody (immunofluorescence) test; distinct pattern specific to the disease is seen
Diagnostic applications in cancer
  • Diagnosis of invasive carcinomas, which present as bright masses with irregular borders composed of fluorescent neoplastic nuclei with surrounding hypofluorescent fibroadipose tissue (Mod Pathol 2014;27:460)
  • In high grade invasive carcinomas, nuclear pleomorphism can be distinguished as irregularly shaped bright dots larger than those observed in well differentiated carcinomas
  • Determines margin status in small breast tumors, thyroid lesions (< 1 cm) and lymph nodes, where the preservation of tissue integrity is important and frozen sections are not feasible
  • Fluorescent in situ hybridization (FISH):
    • Effective for analysis of interphase nuclei (see image below), transcription, genes, chromosomal translocation and small chromosomal aberrations; aids in the diagnosis of multiple myeloma and leukemia
    • Vital tool in selecting a targeted therapy in leukemia by providing reliable biomarker information
    • Immunohistochemistry (IHC) using FISH detects protein overexpression and gene amplification (HER2 status in breast cancer)
    • See also Methods chapter
Molecular / cytogenetics images

Contributed by Ruhani Sardana, M.B.B.S. and Anil Parwani, M.D., Ph.D., M.B.A.
Translocation involving EWSR1 gene at 22q12 (FISH)

Translocation involving EWSR1 gene at 22q12 (FISH)

Contributed by António Polónia, M.D., Ph.D., Ana Caramelo, B.Sc., Catarina Eloy, M.D. and João Vale, M.Sc.
1p/19q codeletion

1p/19q codeletion

Gastric lymphoma

Gastric lymphoma

Lymph node

Lymph node

Applications in research
  • Analysis of intracellular distribution of the nucleus, cell membranes and various intracellular structures, such as cytoskeletal filaments, mitochondria, golgi apparatus
  • Studying living cells and measuring pH, free calcium and NAD(P)H concentration in the cytoplasm
  • Visualizing DNA and RNA sequences in molecular cytogenetics by using fluorescent probes, which bind to segments of nucleic acid showing high degree of sequence complementarity
  • Visualizing molecular processes in relation to bone remodeling in metastasized cancers (J Bone Oncol 2019;17:100249)
  • Monitoring viral attachment process and different steps in the virus replication cycle by analysis of tryptophan emission spectra (Viruses 2018;10:250)
  • Studying various receptor binding affinities of viruses
  • Studying different stages of cell replication, most commonly interphase versus metaphase
  • Suitable for rapid tumor detection due to the possibility of enhancing visibility of nuclei
  • No frozen section related artifacts are present, thus better evaluation of surgical margins
  • Shorter turnaround time
  • Preservation of tissue integrity for routine histological examination and easy detection of adipose tissue (Mod Pathol 2014;27:460)
  • Remarkable morphological correspondence to conventional H&E stained light microscopy
  • Plane of imaging can be precisely chosen without wasting tissue, especially useful for small specimens (Arch Pathol Lab Med 2011;135:255)
  • Signals from fluorescence probes are qualitative and lack accurate quantitative information due to varying permeability of fluorescence dyes
  • Limited repeated measurements due to photobleaching and phototoxicity (Yale J Biol Med 2018;91:267)
  • Requires training in imaging interpretation, especially for black and white images (Mod Pathol 2014;27:460)
Recent advances
  • FISH whole slide imaging
    • Highly sensitive method for interpreting FISH slides with break apart probes, as it can detect a significantly smaller quantity of cells with aberrant or no signals
    • Split signals are easily detected with a better image definition in cells containing MYC rearrangement (Hum Pathol 2013;44:1544)
  • Multiplex immunofluorescence (mIF)
    • Powerful tool for immune profiling to achieve a targeted therapy
    • Fluorescent opal dyes are conjugated with tyramide molecules to produce enzymatic amplification
    • The emergence of slide scanners that can capture fluorescent whole slide images and advancements in autostainers capable of automating mIF protocols helped in measuring several biomarkers (even those infrequently expressed) concomitantly without cross reactivity (Sci Rep 2017;7:13380)
  • Intraoperative real time cancer detection with an integrated lensless fluorescence contact imager to reduce minimal residual disease (MRD) (Biomed Opt Express 2018;9:3607)
  • In vivo optical imaging of cancer cell function and tumor microenvironment (Cancer Sci 2018;109:912)
  • Pathway of metastasis of cancer cells through angiotropism using confocal fluorescence microscopy
  • Intravital microscopy and fluorescent protein based genetically encoded biosensors (Pathol Int 2020;70:379)

Fluorescence microscopy animation

Intro to fluorescence microscopy

Board review style question #1

Which technique is shown in the image above?

  1. Electron microscopy
  2. Enzyme linked immunosorbent assay
  3. Fluorescent in situ hybridization
  4. Immunofluorescence
Board review style answer #1
C. Fluorescent in situ hybridization (FISH) is a diagnostic modality, based on the principle of fluorescence microscopy, that is used to detect both structural (translocation / inversion) and numerical (deletion / gain) genetic aberrations.

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Reference: Fluorescent microscopy applications
Board review style question #2
The principle of fluorescent microscopy is based on which of the following phenomena?

  1. Absorption and radiation
  2. Excitation and emission
  3. Series of glass lenses magnifying the image
  4. Transmission of electrons
Board review style answer #2
B. Excitation and emission. Optical imaging through fluorescent microscopy is based on 2 key spectrums, excitation and emission. It filters out the excited light without blocking the emitted fluorescence, which helps to view the objects that are fluorescent.

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Reference: Fluorescent microscopy applications
Board review style question #3
Which of the following types of microscopes allows sections to be imaged at different depths and a 3D representation to be reconstructed?

  1. Confocal fluorescent microscope
  2. Digital imaging
  3. Electron microscope
  4. Epifluorescent microscope
Board review style answer #3
A. Confocal fluorescent microscope. Confocal microscopes reject out of focus light from the image with a pinhole aperture. By changing the focal plane in a confocal microscope, sections can be imaged at different depths in the specimen and a 3D representation can be reconstructed. This helps in precisely choosing the plane of imaging without wasting tissue; especially useful for small specimens.

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