Technical Guide

GFluorescence Detection (Confocal)

For detecting fluorescence, it is convenient to use a laser with an optical fiber output. If you intend to focus the laser up to the

diffraction limit, use single-mode optical fiber. The infinity-corrected objective lens inside the beam aligner block collimates the light

beam emitted from the optical fiber. In this situation, you can collect the light efficiently if you use an objective lens with an NA that is

larger than that of the optical fiber. You can calculate the beam diameter from the optical fiber NA or the objective lens NA, whichever

is smaller, and the focal length of the objective lens.

The laser is reflected by the dichroic mirror, passes through the objective lens in the stage block, and excites the sample. The excited

fluorescence passes through the objective lens, dichroic mirror, and tube lens block and is focused on the position of the pinhole that

is installed in the pinhole block. This creates the confocal optical system. The light that passes through the pinhole is collimated by

the installed lens and is injected into the PMT module.

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GConnection Example Using an Objective Lens and the Tube Lens Block

An infinity-corrected objective lens is connected to the A10859 tube lens block with a microscope objective lens mount, which has

threads for a M20.32

× 0.706 mm objective lens and a C-mount male thread. Next, the tube lens block is connected to an imaging

camera, such as an ICCD camera. You can adjust the focus using the manual focusing ring on the tube lens block.

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The performance of the confocal optical system is determined by the size of the pinhole. The Airy disk (diameter) is used as the

reference. The following equation is used to calculate the Airy disk. The system magnification is determined by the focal length of the

observation-side objective lens and the focal length of the tube lens block.

If spatial resolution is important, use a pinhole whose size is close to the size of the Airy disk. If you are only measuring fluorescence

intensity, make the pinhole larger than the Airy disk; this will lower the spatial resolution, but it will also increase the signal level.

Airy disk (diameter) = 1.22

× M ×

M: System magnification =

λ : Fluorescence wavelength

NA: NA of the observation-side objective lens

* If the objective lens focal distance on the observation side is unknown, please contact the lens manufacturer.

Focal length of the tube lens block

Focal length of the observation-side objective lens*

λ

NA

ICCD

CAMERA

MICROSCOPE OBJECTIVE LENS MOUNT

*

(COMMERCIALLY AVAILABLE)

OBJECTIVE

LENS

TUBE LENS BLOCK A10859

* Example: Edmund Optics

Microscope objective lens mount

LASER

PMT MODULE

STAGE BLOCK

A10858

C-MOUNT INTERCHANGEABLE

DICHROIC BLOCK A11214

PINHOLE HOLDER BLOCK A11027

OBJECTIVE LENS

(COMMERCIALLY AVAILABLE)

BEAM ALIGNER BLOCK A10760

OPTICAL FIBER (FC CONNECTOR)

OBJECTIVE LENS

(COMMERCIALLY AVAILABLE)

C-MOUNT INTERCHANGEABLE

FILTER BLOCK A11213 (FOR THE CONNECTOR)

C-MOUNT INTERCHANGEABLE

FILTER BLOCK A11213

(FOR THE CONNECTOR)

FILTER BLOCK A10033-xx

ADAPTER BLOCK A10030-01

DICHROIC MIRROR (COMMERCIALLY AVAILABLE)

PINHOLE (COMMERCIALLY AVAILABLE)

TUBE LENS BLOCK A10859-01

C-MOUNT DOUBLE MALE THREAD RING

(SUPPLIED WITH A10859-01)