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.
OPTICAL FIBER (FC CONNECTOR)
BEAM ALIGNER BLOCK A10760
LASER
OBJECTIVE LENS
(COMMERCIALLY AVAILABLE)
OBJECTIVE LENS
(COMMERCIALLY AVAILABLE)
C-MOUNT INTERCHANGEABLE
FILTER BLOCK A11213 (FOR THE CONNECTOR)
PINHOLE HOLDER BLOCK A11027
STAGE BLOCK
A10858
PMT MODULE
ADAPTER BLOCK A10030-01
FILTER BLOCK A10033-xx
PINHOLE (COMMERCIALLY AVAILABLE)
TUBE LENS BLOCK A10859-01
C-MOUNT INTERCHANGEABLE
FILTER BLOCK A11213
(FOR THE CONNECTOR)
C-MOUNT INTERCHANGEABLE
DICHROIC BLOCK A11214
C-MOUNT DOUBLE MALE THREAD RING
(SUPPLIED WITH A10859-01)
DICHROIC MIRROR (COMMERCIALLY AVAILABLE)
TPMOC0217EA
Technical Guide
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.
λ
Airy disk (diameter) = 1.22 × M ×
NA
Focal length of the tube lens block
M: System magnification =
Focal length of the observation-side objective lens*
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.
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.
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.
TUBE LENS BLOCK A10859
OBJECTIVE
LENS
ICCD
CAMERA
MICROSCOPE OBJECTIVE LENS MOUNT *
* Example: Edmund Optics
(COMMERCIALLY AVAILABLE)
Microscope objective lens mount
TPMOC0215EA
21
HAMAMATSU [ HAMAMATSU CORPORATION ]
