使用表面扫描成像（SSI）的共焦显微镜对具有不均匀或粗糙表面的样品进行拉曼成像

共焦拉曼显微光谱仪是分析具有亚微米级高空间分辨率的材料的绝佳选择。这使得能够表征和成像微小成分，如原料药或填充剂、小至1μm的异物以及极薄的层。然而，为了实现高空间分辨率，必须使用具有高放大率的物镜，从而导致极窄的depth-of-field；测量表面上焦点的任何微小偏差都会极大地影响测量光谱的强度。因此，具有不平坦或倾斜表面的样品的成像测量可能导致较差的测量，除非物镜在每个测量点重新聚焦。这导致测量时间非常长。为了在短时间内进行极快的测量，JASCO开发了表面扫描成像（SSI）。SSI用于在测量之前评估样品的表面形状作为载物台的高度信息，并在根据该表面形状信息扫描载物台时执行拉曼测量。使用SSI，即使是具有非常不均匀表面的样品现在也可以被测量，以在所有数据点上给出具有高S/N的良好拉曼光谱。Application Note260-AN-0017 Raman Imaging of Samples with Uneven or Rough Surfacesusing Confocal Microscopy with Surface Scan Imaging (SSI)2/3Application Note Intro d uc t ion A confocal Raman microspectromete r i s an excel l ent choice for ana l ysis of materials with h igh spatial resolution - in the order of su b microns. Th i s makes i t possib l e to cha r acterize and image minute components such as APls or bulk i n g agents and foreign mater i als as small as 1 u m， as well a s extremely th i n layers. However，to ach i eve the high spat i al resolutio n it is necessary to use objective lenses with high magni f ication， r esult i ng in a dept h -of-field that is extreme l y narrow； any slight deviation in the focus at the measurement su r face greatly affects to the intensity of the measured spectrum. As a result ， imaging measurement of a sample wit h ar uneven or a sloping surface may resul t i n poor meas u remen t unless the objective le n s i s r efocused at each measu r ement poi n t . Thi s results in extremely long measurement times. NRS-4100 To make extremely fast measurement in a short time JASCO has developed Surface Scan I maging (SSI). SSl is used to assess the surface shape of the sample as heig h t informat i o n of the stage pr i or to measurement and performs Raman measurement wh il e scanning the stage according to this surface shape informat i on. Using SSl ， even a sample with a very uneven surface can now be measured to give good Raman spectra wit h high S/N at all data points . Keyword s Raman， Su r face Scan Imaging， SSI， Confoca l Raman Imaging C omparison of S SI with C onventional Autofocus Techn i qu es Conventionally， Raman imaging measu r ement of a sample with surface r oughness was performed with autofocus (AF) was carr i ed ou t a t eac h measurement poi n t (figure 1-a)， or XYZ 3D imaging m e asureme n t was carried out (figure 1-b). SSl (f i gu r e 1-c ) calcu l a t es th e sample hei g h t (irregulari t y) i n formation f ro m a multifocus* image and performs imaging measurement wh il e scann i ng t he stage based on this informatio n . Although i t also depends on measurement conditions and sample shape， the time r equi r ed for Rama n imagi n g measurement is greatly shortened compared with the conve n tional m ethod. * Th e multifocus i mag e (all -focus i mage) i s a n i mage obta i ned when the stage is scanned in t h e Z direction to acquire multiple observation images with d i fferent focal poi n ts， and each l ayer that is in focus is extracted and a single in-focus image' i s syn th esi z ed for the en ti r e f i e l d of view. This mult i focus i mage allow the detai l s the entire surface shape of the sample to be identi f i e d prior to measurement. Figure 1. Uneven surfa c e s am p l e me a suremen t. R aman Imag i ng Measu r ement of a Pharmaceu t ica l Table t us i ng S S I Imaging analysis of the uneven par t of the s u rface of a tablet (concave and co n vex difference： approxima t e l y 170 um) was measured u sing SS I . The measuremen t parameters are as fol l ows： Measurement Conditions Instruments NRS-4500 Laser Raman spectrometer Excitation Wavelength 532 nm Objective Lens 20x (long working distance type) Interval 10 um Measurement Points 54x28 points Exposure Time 0.5 second JASCO INC. 28600 Mary's Cour t ， E a s to n ， M D 21601US A T el ： (800) 333-5272 F ax： (410) 822-7526A ppl ic a tio n Lib r ary： ja s c o i n c.com /appli c a t i o ns When m easuremen t i s performed f ollowing the su r face shape by us i ng SSl， a clear Rama n chemical i mage can be obtained because the focus match at each point . By over l apping t h e Raman chemical image on the 3D surface shape i mage， the quant i tat i ve result and shape information of concave-convex part of t h e sample can be checked simultaneouslv. Co nc lusion Using SSI ， Raman imaging measu r eme n t of a sample with sur fa ce u n eve n ness and in c l inat i on can be performed easily a n d quick l y. In addition to t h e tablet measureme n t shown he r e ， SSI make i t easier to a n alyze a wide rang e of samples with s u r f ace roughn e ss inc lu ding r ock and conc r e te， DLC coat in g， r esins， foods， medic i n e s， fertiliz e r gra nu les etc . JASCO INC. 28600 Mary's Cour t ， E a s to n ， M D 21601US A T el ： (800) 333-5272 F ax： (410) 822-7526 A ppl ic a tio n Lib r ary： ja s c o i n c.com /appli c a t i o ns