Fluorescent exciting

For technicians and sales managers!

 

 

In FL exciting the scanner uses fluorescence to generate an image of FOV. The specimen is illuminated with light of a specific wavelength which is absorbed by the fluorophore causing it to emit light (a different color than the absorbed light). The illumination light is separated from the much weaker emitted fluorescence using a spectral emission filter. In this manner, the distribution of a single fluorophore is imaged at a time. In order to make a successful setup may be important to consider the spectral property of the light source. It is also important to know the characteristics of the fluorophores used to label the specimen, because the filter cubes must be chosen to match the spectral excitation and emission characteristics of them.  Mainly components for staining and preparing of the specimen are discussed as well as principles of exciting and imaging of stained specimens.

The following description handles knowledge and principles to understand the components and construction of the Reflector Turret Unit (RTU) and components for fluorescent scan processes.

Because there are very much staining materials (fluorophores), many light filters and light sources, also for special purposes available, it is important to know which combinations are possible and constructive or should be avoided or are impossible.

 

 

Contents

General

Basics and components

            Visible light

            Fluorophore

            Photobleaching

            Filter block

            Characteristics of camera sensor

Principles of exciting and imaging

Define the components

 

General

 

 In Medicine or The Life Sciences, it is often important to differentiate cell compartment like cell membrane, nucleus or pathological statuses like normal or tumor tissue. To do so, fluorescence staining is used. The fluorescent dye can be a small molecule, or protein. To dye the specimen, specific organelle markers or antibodies labelled with fluorophore is used. These dyes are bonding to a special biochemical structure and serves as a marker of this structure. A wide range of fluorophores is available to label these markers and antibodies, by using different fluorophores (stains) for desired structures and staining the tissue, a stained specimen is created. Often the specimen is stained with 3,4 or 5 fluorophores, seldom more.  

 

 

See also:     Epi-Fluorescence Microscopy                                  LINK     (stored)

                     Fluorescence and Fluorescence Applications      (stored)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Basics and components

 

               Visible light

 

Principally it is commonly known that the visible spectrum of light is found in the range from about 380nm to 780nm. The name of the color people learned in his childhood, therefore there are often differences in naming colors, mainly if the clear color crosses over to the next color, e.g. blue to green and so on. To make the color more precise, mainly in technical aspects, we can use the frequency or the wavelength of the color.

Fluorescent scan components are using the wavelength of the light. Because people are working with color names and technique is working with wavelengths, a comparison of both is sometimes helpful.

 

 See also:  Wavelength to Colour Relationship      interactive spectrum of visible light

 

 

 

               Fluorophore

fluorophore (or fluorochrome) is a fluorescent chemical compound that can emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, planar or cyclic molecules with several π bonds. The fluorescence is the emission of light by a fluorophore that has absorbed light. The emitted light has a longer wavelength, and therefore lower energy, than the absorbed (exciting) light. The fluorophore ceases to glow immediately when the light source stops. Wavelengths of maximum excitation and emission are the typical terms used to refer to a fluorophore, but the whole spectrum may be important to consider. Fluorophores are generally used to stain tissues, cells, or materials in a variety of analytical methods, i.e. fluorescent imaging and spectroscopy. See Definitions

 

 

  • A fluorophore is excited by absorbing high energy from a light source of appropriate wavelength and emits low energy and low intensity longer wavelength.

 

Important

True for all fluorophores:   High energy, exciting wavelength is always shorter than the low energy emitted wavelength; the difference is more 10nm.

 

Fluorophores, selection

Fluorophore

Excit

Emiss

DAPI

358

463

Hoechst 34580

392

440

Alexa Fluor 405

401

421

Pacific Blue

404

455

Oregon Green 488

49

526

EGFP

489

511

Alexa Fluor 488

495

520

Calcein

494

514

Alexa Fluor 546

556

572

Rhodamin phalloidin

558

575

SpectrumOrange

554

587

Turbo RFP

553

573

Alexa Fluor 647

653

668

ATTO 647

644

670

DyLight 649

651

673

SpectrumFRed

650

673

Wavelength in [nm]

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

See also:       Fluorophore                                                                                      Wikipedia;

                        Fluorescence Excitation and Emission Fundamentals            (stored)

                        Fluorochrome Peak Excitation and Emission Wavelengths    (stored)

                        Fluorophore table                                                                             (stored)

                        DAPI                                                                                                   Wikipedia

                        Dyes                                                                                                   IDT

                        Spectra Database                                                                             University of Arizona

 

 

 

 

 

 

 

 

               Photobleaching of the specimen

 

PhotobleachingIn fluorescent microscopy, the phenomenon of photobleaching (sometimes termed fading) occurs when a fluorophore loses the ability to fluoresce permanently. Photobleaching is provoked by non-specific reactions between the fluorophore and surrounding molecules caused by the excitation light. Some fluorophores bleach quickly after emitting only a few photons, while others that are more robust can bear thousands or millions of cycles before bleaching. Loss of activity caused by photobleaching can be controlled by reducing the intensity or time of light exposure.

 

ˇ         The scan quality of photobleached specimen is (drastically) reduced.

ˇ                                 Fully bleached specimen is unusable in fluorescent scan procedures.

 

Responsibilities for bleaching

 

ˇ             Light and heat sensitivity.      Because fluorophores are sensitive to light and heat, the specimen should be stored in a dark and cool surrounding (e.g. in a box of the refrigerator).

ˇ             Sunlight sensitivity.                 Never expose the stained specimens to sunlight, this would increase bleaching drastically.

ˇ             Exposure time sensitivity.       Minimize the exposure time of the Excitation light by switching it off after the image is taken or remove the filter block from the exiting path.

ˇ                                                                                                                   Use the live view for adjustments as short as possible.

ˇ             Exciting power sensitivity.      Decrease the exciting power and time as possible.

 

See also:       Photo bleaching      (Wikipedia)

                        Photo bleaching      (Florida State University)

                        Photo bleaching      (stored)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

               Filter block

 

Optical filters are used to selectively transmit light in a range of wavelengths while reflecting wavelengths outside the defined range. They can usually pass long wavelengths only (longpass), short wavelengths only (shortpass), or a band of wavelengths, blocking both longer and shorter wavelengths (bandpass). Dichroic mirrors (beamsplitters) are specialized filters which are designed to efficiently reflect excitation wavelengths and pass emission wavelengths. The filter suitable for the exciting wavelength and the filter fitted for the emitting wavelength as well as the dichroic mirror are mounted in a filter block.

The filters of the filter block are combined for a special stain. In other words, every stain (fluorophore) has its optimal exciting wave length and the appropriate optimal emitting wavelength. For these wavelengths the filter block components are combined. This means also, that every stain has its own filter block, but one filter block may serve several analogous fluorophores.

The filter block filters the exiting light, arriving from the excitation light source, and the dichroic mirror directs it via the objective to the stained specimen and excites there the fluorophore. The excited fluorophore of the specimen’s FOV emits light in a longer wave length. The emitted light is collected by the objective and passes through the dichroic mirror and the emission filter to the camera.

Single band filters will be filtering one exciting wave length (range) and will be filtering the adequate emission wavelength (range).

 

Advantage

The exciting wavelength can be filtered from a white light source.

 

Disadvantage

If another exciting wavelength is required, the filter set has to be changed physically and this is time consuming in relation to the scan procedure.

 

 

 

Exciting and emission wavelength

On filters and other components principally the center wavelength and the guaranteed bandwidth is defined.

The bandwidth is divided by two and one half number is subtracted from, while the other half is added to the center wavelength.

 

  • The value 438/24 (SpAqua) means, the center wavelength is 438nm and the bandwidth is 24nm.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Standard single band filter sets

Filter

Exciting

Emission

Art. number

Comment

SpAqua

438/24

483/32

SPAQUA01

ZPS

Cy3

531/40

593/40

CY301

ZPS

DAPI 2

387/11

447/60

DAPI02

ZPS

Cy5

628/40

692/40

CY501

ZPS

DAPI 1

377/50

447/60

DAPI01

ZPS

FITC

485/20

524/24

FITC01

ZPS

LF635

640/14

676/29

LF63501

LF;  ZPS

mCherry

578/21

641/75

MCHERRY1

LED; ZPS

SpGold

534/20

572/28

SPGOLD01

ZPS

SpGreen

494/20

527/20

SPGREE01

ZPS

SpOrange

543/22

586/20

SPORAN01

ZPS

SpRed

586/20

628/32

SPRED01

ZPS

Because the Article number is very long; it is truncated in the table, only the relevant part is shown.

The redundant prefix   "HP-FLT-SR02-" is used for all article numbers.

Example:  SPAQUA01 read as  HP-FLT-SR02-SPAQUA01.

LED=  LED based light sources; LF= LASER Filter

Sp= Spectrum; ZPS= Zero pixel shift

Other filters are available  upon request, according to requirement specification

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The following link may help you to find possible as well as impossible combinations.

 

ˇ      “Matching Fluorescent Probes with Nikon Fluorescence Filter Blocks”; interactive

 

 

 

 

 

Because the filterblock has to be changed if the exciting wavelength will be changed, and this procedure is time consuming related to the scan process, multiband filters are created. 

 

 

Multiband

Quadband_Semrock.gif

Multiband filters combining more excitation and adequate emission wavelengths inside of one filter set.

In Pannoramic scanner types mainly quad band filters are used as multiband filters.

 

Advantage

The filter block has to be changed only, if an exciting wavelength is required which is not included in the quad band set.

This way the scan procedure is done much faster because the filter block has to be changed less often.

 

 

Disadvantage

The exciting wavelength cannot be filtered from a white light source. Every exciting light wavelength has to be created separately and will be switched on or off as required.

This construction makes the exciting light source more expensive.

 

 

Pannoramic scanners using mainly BrightLineŽ Multiband Fluorescence Sets from Semrock, therefore, this will be used as example also.

 

 

 

 

 

 

 

 

 

 

 

 

Parameters of filter set

 

DA/FI/TR/CY5-A-000

Name

Exciting

Emission

Dichroic

Art. number

DAPI

387

440

410

HP-FLT-SR07

-QUAD02

FITC

485

521

504

TRITC

559

607

582

Cy5

649

700

669

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Frequently used fluorophores in conjunction with quadband filters

 

 

Fluorophores and filter parts

Fluorophore

Excit

Emiss

Part of Quad band

DAPI

358

463

DAPI

392/432/409

Hoechst 34580

392

440

Alexa Fluor 405

401

421

Pacific Blue

404

455

Oregon Green 488

488

526

FITC

474/515/493

EGFP

489

511

Alexa Fluor 488

495

520

Calcein

494

514

Alexa Fluor 546

556

572

TRITC

554/595/573

Rhodamin phalloidin

558

575

SpectrumOrange

554

587

Turbo RFP

553

573

Alexa Fluor 647

653

668

Cy5

635/730/652

ATTO 647

644

670

DyLight 649

651

673

SpectrumFRed

650

673

Wavelengths are in [nm]

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

See also:       Filter set details       Semrock (stored)

 

 

 

 

 

 

               Characteristics of camera sensor

 

 

Scan camera

Name

Type

Adimec Q-12A180

mono/color

PCO.edge 5.5Mp

mono

PCO.edge 4.2Mp

mono

AxioCam MRm Rev.3

mono

GS3-U3-51S5M

mono

CIS VCC-F52U25CL

color

CIS VCC-FC60FR19CL

color

Stingray F146C

mono/color

Marlin F146C

mono/color

Hitachi

color

Sony DFW710

color

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Further information about the camera can be found in the chapter Scan cameras

 

 

 

 

 

 

 

 

 

Principles of exciting and imaging

 

White_light_exchange_filter.gifWhite light source

 

Traditionally, exciting of specimens is done from a white light source with exciting wavelength range of 350nm to 800nm.

By using wavelength filters, the required exciting wave length (range) is filtered from the white light and this is used to excite the fluorophore via the filter block and the objective.

Generating the exciting wavelength by filtering from white light requires a filter block for each fluorophore separately, this means, if the exciting wavelength is changed, the filter block has to be exchanged also.

Because the emitted light is very low in intensity, the exposure time of the camera is increased, in relation to the brightfield image and if there e.g. 4 stains are used, 4 images of the same FOV is taken, each with another filter block.

The filter block has to be moved every time into the image path. Therefore, scanning of a stained FOV may take several seconds; a small tissue of several 100 FOVs may take 10 … 20minutes.

 

ˇ         As discussed before, the scan time of a FOV is the sum of exposure time for each FOV and the time for the filter block exchange.

 

To increase the scan speed, the time for filter exchange is minimized by using multiband filters in the filter block.

 

Light sources, that creating white light is the “X-Cite type Series”, HXP 120 and the more cost effective “SOLA-SM-II-Light-Engine”

If the light source creates white light, only single band filters can be used. The light wave length filters in the filter cube are filtering out all unused light wavelengths and only the desired, special wavelength will be used to excite the specimen.

The filter block creates a single monochrome wavelength from the white light for exciting the specimen. Each required wavelength requires also a separate filter block to create the desired monochrome light wavelength.

For this purposes, the turret unit has 10 positions and is able to handle 9 filter blocks for fluorescent scan operations.

 

 

See also:       Introduction to Fluorescence Filters       Semrock

 

 

 

 

 

 

 

 

Monochrome light excitation

 

The principle of monochrome excitation includes the creation of exciting wavelengths separately. The light source (light module) creates the light wavelength as required for the fluorophore.

Monochr_excitation.gif

By using multiband filters (e.g. Quad band filters), 4 exciting wavelength can be filtered with the same filter block, without moving the filter block. This way FOV scan time is saved, because there are no mechanical movements of the filter block.

 

 

Light source, which create monochrome light is the “Lumencor SPECTRA light engine”.

The main difference is, that the light source creates the required monochrome light wavelengths separately and these can be switched on or off as required very quickly.

Because the exciting light is monochrome, multi channel filters can be used and so, the movement of the filter blocks is practically eliminated.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

               Exciting light source

 

To excite the fluorophore it will be illuminated with very high intensity light by using a special wavelength, the exciting wavelength of the fluorophore.  The required wavelength of the light is supplied by special excitation lamps.

 

See also:  Fundamentals of Metal Halide Arc Lamps           Link

 

 

 

 

 

 

HXP 120

 Mainly used in:     MIRAX SCAN and MIRAX MIDI

 

Metall halide fluorescence light source. Connected via liquid light guide, Vibration free with integrated shutter, software controlled.

 

See also:     Light Electronics Jena          (LEj home page)

                     LQ-HXP 120 Manual            (stored)

 

 

 

ˇ        By calculating of components, the emitted wavelengths of the light source is important!

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

X-Cite 120

             Creation of white light

 

 

In Pannoramic configurations 2 types are used.

 

 

See also:       Light sources main page

                        Precautions (stored)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

By calculating the parameters of components, the emitted excitation wavelengths of the light source are important!

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SOLA-SM-II-Light-Engine

 

 Used in:  P250, SCAN and MIDI

 

 

 

 

 

 

 

Sola-SM-II-Light-EngineŽ offers a more cost-effective solution in relation to the HXP-120 Light SourceŽ or to the Lumencor SPECTRA light engineŽ and may be used in Fluorescent scan sessions of any scanner type.

  • Because the Light engine emits white light, only single band filters can be used.

 

 

 

See also:       Users manual

                        Instruction manual

                        Filter Set Recommendations Semrock

 

 

 

 

 

 

 

 

 

 

 

 

By calculating of components, the emitted excitation wavelength of the light source is important!

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Lumencor SPECTRA light engine

 

 Used in:  P250, SCAN and MIDI

 

 

 

 

Early delivered Lumencor  SPECTRA

Color

channel

Bandpass /width

Range

[nm]

[nm]

Violet

386/23

375 ~ 398

Blue

438/24

426 ~ 450

Cyan

485/20

475 ~ 495

Teal

512/25

499 ~ 525

Green

550/88

506 ~ 594

Yellow

N/A

N/A

Red

650/13

643 ~ 657

nIR

Not implemented

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Real values see Certificate of conformance for connected component.

 

 

 

 

 

 

 

 

 

 

 

New spectrum of Lumencor SPECTRA

Color

channel

Bandpass /width

Range

[nm]

[nm]

Violet

390/22

379 ~ 401

Blue

438/29

424 ~ 453

Cyan

475/34

462 ~ 488

Teal

513/22

502 ~ 524

Green

542/33

526 ~ 555

Yellow

575/35

558 ~ 593

Red

631/28

617 ~ 645

nIR

Not implemented

 

Values for orientation only. Real values see Certificate of conformance for connected component.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

By calculating components, the emitted wavelength of the light source is important!

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Define the components

 

This part should help you to select the right components for powerful FL scanning equipment, beginning from different circumstances.

 

 

Fluorophore, Filter set and Light source

 

As discussed before, the wavelengths ranges of the components should be matched optimally to reach best possible scan quality; this means:

 

The wavelength range intersection of the

  • fluorophore's exciting wavelength,
  • the wavelength of the exciting filter and
  • the wavelength of the exciting light source

should be as much as possible.

 

The wavelength range intersection of the

  • fluorophore's emission wavelength range,
  • the emission wavelength range of the filter and
  • the wavelength spectrum of the camera

should be as much as possible.

 

New system

 

Starting with the fluorophores

  • The user should provide a list of frequently used fluorophores.
  • Make a table with the exciting and the emission wavelength
  • decide the light source, white light or monochrome excitation
  • decide the filters; single band, multi band and exciting and emission spectra
  • Define the scan camera type

 

  • While defining the components for the system some modifications may be required.
  • If in the list of the user's fluorophores are items, that can not be excited with the planned light source or can not visualized with the planned camera, discuss these items with the user.
  • Keep in mind, that the components of the system are expensive, so lifetime and maintenance costs may be relevant.

 

 

Existing system

 

In an existing system we assume, the filter blocks, the excitation light source and the camera are existent.

 

  • Compare the emission spectrum of each filter block with the spectrum of the camera
  • Make a table of emission spectrum and exciting spectrum of the  filter blocks.
  • find useable fluorophores depending on the spectrum of the light source and the spectrum of the filter block's filters.

 

 

 

Links

SearchLightTM Introduction                                  Semrock

SearchLightTM Analysing and plotting tool        Semrock

 

The SearchLightTM Analysing and plotting tool can be used to compare exciting and emission wavelengths of filters (sets), fluorophores and light sources.

 

 

Further links

To find filters, fluorophores, Light engines and optimizing component selection for the user's requirements and also for planning of fluorescent scan systems, the following links may help.

http://www.spectra.arizona.edu/

https://www.micro-shop.zeiss.com/index.php?s=91531866c91b70&l=en&p=de&f=f&a=f

https://www.semrock.com/setdetails.aspx?id=2983

https://www.chroma.com/products/complete-filter-sets