激光扫描显微镜是一种使用激光器作为光源对目标物体进行成像的光学显微镜。
根据用途和探测器配置,激光扫描显微镜可分为多种类型,因此其内部结构和工作原理也会彼此不同。
这里,我们以典型的激光共聚焦显微镜为例,介绍其工作原理。
扫描激光的方法和光学系统的配置取决于每种类型的显微镜。激光共聚焦显微镜的一个独特特点是设置在焦点处的针孔。针孔消除了测量不需要的光,因此只会检测到非常接近样品焦点平面的荧光。由于这种结构,激光共聚焦显微镜允许在深度方向以优异的光学分辨率和分辨力进行测量。
滨松光子学株式会社开发和制造大量关键设备,这些设备对于激光扫描显微镜的操作和性能至关重要。
光电检测器是激光扫描显微镜的核心,与采集图像的质量直接相关。在众多光电检测器中,具有出色灵敏度和噪声特性的光电倍增管 (PMT) 和通常称为硅光电倍增器或 Si-PM 的多像素光子计数器 (MPPC) 广泛用于采集具有更高质量的显微图像。
滨松光子学株式会社开发和制造 MEMS(微机电系统)振镜,与广泛用作扫描振镜的电流振镜相比,具有许多优势。这些优点包括超小型尺寸、低成本和低功耗。现在,使用单个 MEMS 振镜设备即可实现二维扫描,这样可以轻松缩小设备尺寸,并大幅加快设备运行。
用于激光扫描显微镜的探测器必须在灵敏度和噪声方面表现出卓越的特性。这很重要,因为激光扫描显微镜的基本测量原理限制了入射到探测器上的光量。
例如 1 红外激光共聚焦显微镜:针孔减少了入射到探测器上的光量。
例如 2 多光子显微镜:发生多光子吸收现象的概率极低,因此光量有限。
探测器有多种类型,具有不同的功能,因此必须选择最符合显微镜规格的最佳探测器。
这些照片显示了使用不同的 PMT 作为激光扫描显微镜的探测器观测到的相同样品的图像。
与使用多碱光阴极面 PMT 采集的左侧图像相比,使用 GaAsP 晶体光阴极面 PMT 采集的右侧图像显示出清晰和明显的对比度。正如此示例表明,即便使用 PMT 等高灵敏度探测器,所获取的图像也会根据其光阴极面类型而有很大不同。
滨松光子学株式会社提供广泛的探测器阵容,让您可以选择最佳探测器,以满足您的测量标准和条件,例如光量、波长、样品厚度、扫描速度和冷却方法。
左图:使用多碱光阴极面 PMT 采集的图像
右图:使用 GaAsP 光阴极面 PMT 采集的图像
※样品:小鼠细胞
多光子显微成像技术是一种用于观测紫外线至可见光区域荧光的技术,通过同时吸收两个光子激发荧光分子时,便会出现这种荧光。
共同荧光测定中激发光的波长短于荧光波长。然而,多光子光谱仪在近红外区域使用波长长于荧光波长的激发光。由于近红外光比可见光更容易通过物体,因此它可以提供关于物体内深层部分的信息,并且还可以减少物体内部的散射和背景噪声的影响。此外,近红外光的能量低于可见光和紫外光,从而最大限度地减少对细胞的损害。
・成像深度:距脑表面 500 μm
・成像范围:3 mm × 3 mm
通过使用高灵敏度光电倍增管(相当于滨松 H15460-40)检测具有高信噪比的信号来获得图像。这种光电倍增管还具有宽受光面,支持在宽视野中观测深部,这是多光子显微成像技术的一个独特特点。
图像来源:日本理化学研究所脑科学中心 (CBS) Masanori Murayama(博士)
产品类别 | 图像 | 产品名称 | 特点 |
---|---|---|---|
探测器 | 光电传感器模块 H15460-40 | 大受光面:14 mm² | |
光电传感器模块 H7422A-40 | 内置电子制冷器:最大限度地减少热噪声 | ||
MPPC 模块 C13852 系列 | 小巧轻便的装置,在可见光波段具有灵敏度 | ||
MPPC 模块 C14456 系列 | 小巧轻便的装置,在可见光到近红外波段具有灵敏度 | ||
光学组件 | MEMS 振镜 S13989-01H | 通过激光反射进行二维扫描 | |
LCOS-SLM X15213 系列 | 用于像差校正的相位控制(波前控制) |
FLIM 是一种用于测量物体发射的荧光的衰减时间(寿命)的技术。
FLIM 通过时间分辨测量来测量每个荧光分子独有的荧光寿命,这样可以为用户提供比常规荧光测定更多的信息。用于 FLIM 的探测器需要具有高速响应和高灵敏度,因为它们必须测量在极短时间内发生的荧光强度的微小变化。
产品类别 | 图像 | 产品名称 | 特点 |
---|---|---|---|
探测器 | HPD | 高速响应 高时间分辨率 |
该技术用光刺激样品并实时观测其反应。主要用于测定生物样品。由于在光刺激测量中使用多个用于激发和刺激的激光器,因此必须创建能够在所需时间准确检测到测量光的光学设计。
具有门控功能的光电传感器模块非常适合作为设备安装在使用光刺激的显微镜中,因为其门控操作可以通过电控制来匹配光探测的时序。
此外,通过使用 LCOS-SLM 波前整形技术的光学系统将激光光束分成多个光束,多个点可以通过同一个激光器的光同时刺激。
产品类别 | 图像 | 产品名称 | 特点 |
---|---|---|---|
探测器 | 光电传感器模块 H12056-40 | 与门电路集成的紧凑型装置 最大输出信号电流:40 µA |
|
光电传感器模块 H11706-40 | 与门电路集成的紧凑型装置 最大输出信号电流:2 µA |
||
MPPC 模块 C13852 系列 | 小巧轻便的装置,在可见光波段具有灵敏度 | ||
MPPC 模块 C14456 系列 | 小巧轻便的装置,在可见光到近红外波段具有灵敏度 | ||
光学组件 | MEMS 振镜 S13989-01H | 通过激光反射进行二维扫描 | |
LCOS-SLM X15213 系列 | 用于生成多点的相位控制(波前控制) | ||
波前整形器 C15789 系列 | 内置 LCOS-SLM 光模 用于生成多点的相位控制(波前控制) |
由于对荧光蛋白质发射的较长波长荧光的测量需求日益增加,这种多色测量技术最近成为备受瞩目的焦点。多色测量可捕获从可见光到近红外的广泛光谱范围的信息,使其成为一种有望揭示前所未有的现象的方法。
具有宽光谱响应范围且对近红外区域也具有高灵敏度的光电检测器是有效的。
产品类别 | 图像 | 产品名称 | 特点 |
---|---|---|---|
探测器 | 光电倍增管 R13456 | 在近红外区域具有高灵敏度 | |
光电倍增管 R10699 | 从可见光到近红外区域的宽光谱响应范围 | ||
MPPC 模块 C13852 系列 | 小巧轻便的装置,在可见光波段具有灵敏度 | ||
MPPC 模块 C14456 系列 | 小巧轻便的装置,在可见光到近红外波段具有灵敏度 |
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