Schulung Mikroskopie III engl - Hund · Fluorescence Microscopy? • Brightfield: „Everything“ is observed • Staining: One Step more specific • Phase contrast: Also full-field

Outline

• Fluorescence Microscopy?

• History

• Fluorescence: Three Mechanisms

we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008

• Fluorescence: Three Mechanisms

• The Fluorescence Microscope

• LED Incident-Light Fluorescence (LED-AFL)

• Examples

Fluorescence Microscopy?

• Brightfield: „Everything“ is observed

• Staining: One Step more specific

• Phase contrast: Also full-field


• Observation of only very specific properties?

• Solution: Specific staining, specific detection!

History

• 1852: Fluorescence phenomenon discovered

• Early 20th century: First applications in microscopy

• FL microscopes: „complex, difficult to operate,


• FL microscopes: „complex, difficult to operate, expensive“

• Since 1960: Xe/Hg high-pressure lamps, specific filter systems, fluorochromes

• Recently: Innovations through high-power LEDs in the UV/VIS

Fluorescence

1852: Fluorescence as a Phenomenon(Sir George Gabriel Stokes (1819 - 1903))

Excitation

Fluorescence


Quelle: www.nndb.com

E

Fluorescence

Fluorescence: Mechanisms

Primary Fluorescence (Autofluorescence)


Pollen Grains (365 nm)


Secondary Fluorescence (Staining)


Yeast (Mykoval)


Immunofluorescence


Coccobacilli (FITC)


Advantages of Fluorescence Microscopy

Bright structures

on a darkbackground


background

Characteristiccolours


Analogy: Astronomy

Bright structures

on a darkbackground


background

Characteristiccolours

Constellation:Microscopium


Advantages of Fluorescence Microscopy

• Unambiguous detection

• Simple technique


• Suitable for routine applications

• No particular training required

The Fluorescence Microscope

Incident-Light Fluorescence („classical“)



Emission Spectrum HBO Lamp



Filter Set: Transmission curves



Classical AFL Microscope

(1) Mercury high-pressure

(HBO) lamp

(2) Power supply(1)(3)

(4)


(3) Light barrier switch

(4) Filter set changer(2)

(4)


Classical AFL Microscope

• HBO lamp

• Special lamphouse,

high-voltage supply


• Warm-up necessary

• Lifetime: 200 h

• Burner: ~ 120 €

LED Incident-Light Fluorescence

Advantages of LED Fluorescence

• Monochromatic

• Low power consumption: some watts

• No infrared radiation emitted


• No infrared radiation emitted

• Available from infrared to ultraviolet

• Lifetime > 5.000 h (at nominal current)

• Price: < 100 €, VIS: < 10 €


Light Source: LED

To Eyepiece

(1) Excitation filter


Specimen

Objective

(1) Excitation filter

(2) Beamsplitter

(3) Barrier filter


LED Spectra (Luxeon)



LED-AFL Unit



LED-AFL Unit: Adaptation


Example: Mycoses

Fluorochrome Mykoval (Filter Set G)

• Exclusive for Helmut Hund GmbH

• Binds to cellulose and chitin

• Marks fungi and yeast


• Marks fungi and yeast

• Simple to apply, lasts for years

• High diagnostic reliability: immediate start of

therapy possible

• Cultivation in most cases unnecessary

Example: Mycoses

Mykoval can be applied with

• Native specimens: skin, hair, nails, smears

• Histologic specimens, combinations with other stainings possible

• Rapid diagnoses in surgery


• Rapid diagnoses in surgery

• Body fluids, stool

• Gynecology: yeast cultures on mucous membranes

• Mikrospores in stool

Example: Mycoses

Trichphyton rubrum in skin scales

Objective 10:1


Objective 10:1

Example: Fungi, Spores

Mold Fungus

Objective 40:1


Objective 40:1


Mold Fungus


Adhesive-film specimen

(indoor sample)


Discrimination Cellulose Fibers



Comparison Brightfield - Fluorescence












Brightfield, Phase Contrast, Fluorescence








Example: Yeast

Staining: Mykoval (Filter Set G)


buds

Example: Staphylococci

Staining: FITC (Filter Set D)


E. Coli

Example: E. coli

Staining: DAPI (Filter Set A)


Example: Mycobakteria (Tb)

Staining: Auramine + Thiazine Red (FS D)


Example: STED-Microscopy

STED: Stimulated Emission Depletion


Source: MPI Biophys. Chemie

Example: STED-Microscopy

STED: Stimulated Emission Depletion


Human Nerve Cell: Filaments

STED: Resolution increased by factor 12!

Quelle: MPI Biophys. Chemie

Example: FISH

FISH: Fluorescence In-Situ Hybridization


Chromosomes from female human erythrozytes

Red: DNA

Green: Alu Sequences

Example: TIRF

TIRF: Total Internal Reflection Fluorescence


Summary

Fluorescence Microscopy:

• Sensitive, highly specific

• Mykoval: Staining for fungi/yeast


• Fluorescence with LEDs:- very simple application- inexpensive technology- small follow-up costs

Documents

Schulung Mikroskopie III engl - Hund · Fluorescence Microscopy? • Brightfield: „Everything“ is observed • Staining: One Step more specific • Phase contrast: Also full-field