Laser scanning microscope

A Laser scanning microscope is a type of optical microscope for imaging a target object using a laser as the light source.

Principle of laser confocal microscope

There are various types of Laser scanning microscopes depending on the application and detector configuration, so their internal structure and operating principle differ from each other.
Here, we introduce the principle of a typical laser confocal microscope as an example.

Laser light strikes a target object (sample) via scanning mirrors.
Fluorescence emitted from the sample is directed to a pinhole.
The scattered light is reduced by the pinhole placed at the focus point and is measured by the detector.

Role of the pinhole in fluorescence measurement

The method of scanning the laser light and the configuration of the optical system differ depending on each type of microscope. One distinct feature of laser confocal microscopes is the pinhole placed at the focus point. The pinhole eliminates light unnecessary for measurement so that only fluorescence very close to the focal plane of the sample can be detected. Due to this structure, laser confocal microscopes allow measurements with excellent optical resolution and resolving power in the depth direction.

Development of key devices for Laser scanning microscopes

Hamamatsu Photonics develops and manufactures a large number of key devices that are essential for Laser scanning microscope operation and performance.

Photodetectors

Photodetectors are the core of Laser scanning microscopes and are directly linked to the quality of the acquired image. Among photodetectors, photomultiplier tubes (PMT) with excellent sensitivity and noise characteristics and multi-pixel photon counters (MPPC) which are often called silicon photomultipliers or Si-PM are widely used to acquire microscopic images with ever higher quality.

Features of photodetectors

High sensitivity: Boosts image quality and resolution of acquired images.
Low noise: Boosts image quality of acquired images.
Wide dynamic range: Boosts image quality of acquired images.

Optical components (mirrors)

Hamamatsu Photonics develops and manufactures MEMS (Micro-Electro-Mechanical Systems) mirrors that offer many advantages over Galvano mirrors which have been widely used as scanning mirrors. These advantages include a subminiature size, low cost and low power consumption. Two-dimensional scanning is now possible with a single MEMS mirror device, which will make devices easy to downsize and drastically speed up equipment operation.

Features of optical components (mirrors)

Miniature size: Helps shrink the equipment size.
High-speed operation: Trims the image acquisition time.
Two-dimensional scanning: Does two-dimensional scanning with a single device.

Selecting detectors

Detectors for Laser scanning microscopes must exhibit excellent characteristics in sensitivity and noise. This is important because the amount of light incident on the detector is limited due to the basic measurement principle of Laser scanning microscopes.

Ex. 1 Laser confocal microscope: The pinhole reduces the amount of light incident on the detector.
Ex. 2 Multiphoton microscope: The probability that the multiphoton absorption phenomenon will occur is extremely low so the amount of light is limited.

There are various types of detectors having different functions, so it is essential to select the optimal detector that best matches the microscope specifications.
The photos show images of the same sample observed using different PMTs serving as the detector for a Laser scanning microscope.
Compared to the left image acquired using a multialkali photocathode PMT, the right image acquired using a GaAsP crystal photocathode PMT shows clear and distinct contrast. As seen from this example, even when using a high sensitivity detector like a PMT, the acquired image will greatly differ depending on its photocathode type.
Hamamatsu Photonics offers a broad lineup of detectors that allow you to select the optimal detector to meet your measurement criteria and conditions such as the light level, wavelength, sample thickness, scanning speed, and cooling method.

Left: Image acquired using multialkali photocathode PMT
Right: Image acquired using GaAsP photocathode PMT
※Sample: Mouse cells

Measurement techniques and recommended products

Multiphoton microscopy

Multiphoton microscopy is a technique for observing fluorescence in the UV to visible region that occurs when fluorescent molecules are excited by simultaneously absorbing two photons.
The wavelength of the excitation light in common fluorescence measurement is shorter than the fluorescence wavelength. However, multiphoton spectroscopy uses excitation light in the near-infrared region, which has a wavelength longer than the fluorescence wavelength. Since near-infrared light passes more easily through an object than visible light does, it can provide information on deep portions within an object and also reduces effects from scattering and background noise inside the object. Furthermore, the energy of near-infrared light is lower than that of visible and UV light, thus minimizing damage to cells.

■ How it differs from single-photon excitation microscopy

Imaging example: Observation of deep mouse brain

･Imaging depth: 500 µm from brain surface
･Imaging range: 3 mm × 3 mm

The image was obtained by detecting signals with a high signal-to-noise ratio using a high sensitivity photomultiplier tube (equivalent to Hamamatsu H15460-40). This photomultiplier tube also has a wide photosensitive area so that observation of deep portions, which is a unique feature of multiphoton microscopy, is possible in a wide field of view.

Image courtesy：RIKEN Center for Brain Science(CBS) Masanori Murayama (Ph.D.)

Recommended products for multiphoton microscopy

Products category	Products name	Features
Detectors	Photomultiplier tube module H15460-40	Large photosensitive area: 14 mm sq.
	Photomultiplier tube module H16722-40	Built-in thermoelectric cooler: Minimizes thermal noise
	MPPC modules C13852 series	Compact and lightweight unit with visible light sensitivity
	MPPC modules C14456 series	Compact and lightweight unit with visible to near-infrared sensitivity
Optical components	MEMS mirror S13989-01H	Two-dimensional scanning by reflection of laser light
	LCOS-SLM X15213 series	Phase Control (Wevefront control) for aberration correction

Fluorescence lifetime imaging microscopy (FLIM)

FLIM is a technique for measuring the decay time (lifetime) of fluorescence emitted from an object.
FLIM measures the fluorescence lifetime unique to each fluorescent molecule by time-resolved measurement, and in this way gives users more information than is possible just from conventional fluorescence measurement. The detectors used for FLIM are required to have both high-speed response and high sensitivity since they must measure small changes in fluorescence intensity occurring within an extremely short time.

Recommended products for FLIM

Products category	Image	Products name	Features
Detectors		HPD	High-speed response High time resolution

Photostimulation measurement

This technique stimulates a sample with light and observes its reaction in real time. It is mainly used for measuring biological samples. Since multiple lasers for excitation and stimulation are used in photostimulation measurement, it is essential to create an optical design that accurately detects the measurement light at the required timing.

Photomultiplier tube modules with a gate function are ideal as devices that mount in a microscope utilizing photostimulation since their gate operation can be electrically controlled to match the timing of light detection.
Moreover, by splitting a laser beam into multiple beams with an optical system using LCOS-SLM wavefront shaping technology, multiple points can be simultaneously stimulated by light with a single laser unit.

Recommended products for photostimulation measurements

Products category	Products name	Features
Detectors	Photomultiplier tube module H12056-40	Compact unit integrated with a gate circuit Maximum output signal current: 40 µA
	Photomultiplier tube module H11706-40	Compact unit integrated with a gate circuit Maximum output signal current: 2 µA
	MPPC modules C13852 series	Compact and lightweight unit with visible light sensitivity
	MPPC modules C14456 series	Compact and lightweight unit with visible to near-infrared sensitivity
Optical components	MEMS mirror S13989-01H	Two-dimensional scanning by reflection of laser light
	LCOS-SLM X15213 series	Phase control (wavefront control) for generating multiple point

Multicolor measurements

This technique is recently the focus of a huge amount of attention due to increasing needs for measuring longer wavelength fluorescence emitted from fluorescent proteins. Multicolor measurements capture information over a broad range of the spectrum from visible to near infrared, making this a promising way to reveal phenomena that have not been seen before.

Photodetectors with a wide spectral response range that is also highly sensitive in the near-infrared region are effective.

Recommended products for multicolor measurements

Products category	Products name	Features
Detectors	Photomultiplier tube R13456	High sensitivity in the near-infrared region
	Photomultiplier tube R10699	Wide spectral response range from visible to near-infrared
	MPPC modules C13852 series	Compact and lightweight unit with visible light sensitivity
	MPPC modules C14456 series	Compact and lightweight unit with visible to near-infrared sensitivity

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