第1章 組織學實驗方法
Chapter 1 Histological experimental method

（一）如何正確使用顯微鏡觀察組織標本

1．對照圖片，結合實物，熟悉顯微鏡的構件。

2．將顯微鏡輕輕地移至觀察者的前方，使鏡座的后緣距實驗臺邊緣大約10 cm。

3．旋開顯微鏡電源開關，并使光線亮度適當。

4．旋轉物鏡轉換器，將10倍物鏡旋至鏡筒下方，對準載物臺中央的孔。

5．打開聚光鏡孔徑光闌，可見光線經聚光器→載物臺中央的孔→10倍物鏡→目鏡。

6．注意身體坐姿，睜開雙眼，把觀察注意力集中于顯微鏡的視場內，可見兩個不完全重合的視場光斑，雙手推移兩目鏡，使兩光斑合二為一。

7．按照實習指導和切片標本目錄，從標本盒內取出要觀察的標本，進行肉眼觀察，初步了解該標本的大小、形狀和染色等。

8．將載玻片放在載物臺上，蓋玻片朝上，標簽位右側，用切片夾夾好。旋轉推進器移動旋鈕，將載玻片上的組織標本移至物鏡下方。調節電源開關，將視野亮度調至適中。在右手旋轉推進器移動旋鈕移動切片的同時，左手慢慢地旋轉粗調旋鈕和微調旋鈕聚焦（先旋粗調旋鈕，后旋微調旋鈕）。因推進器位置在不同顯微鏡可能不同，左右手分工可能互換。

9．顯微觀察標本常規是從低倍放大開始，這樣視場大，有利于對標本進行全面觀察。使用低倍物鏡時，觀察者左手轉動粗調旋鈕，使載物臺緩慢上升或下降，同時，觀察者用雙眼從目鏡中觀察視場，當見到組織標本像輪廓時，轉動微調旋鈕直至組織標本像清晰。

10．當要進一步放大觀察時，則直接旋轉物鏡轉換器，將更高倍物鏡（如4倍轉成10倍，或10倍轉成40倍）旋至鏡筒下方。此時，只需轉動微調旋鈕調焦（必要時升降聚光器或調節聚光鏡光闌的孔徑），便可獲得更清晰的進一步放大的標本像。在不同切片和不同放大倍數下觀察時，應注意隨時調節亮度，使之最適宜。

11．移動切片觀察時，要注意片中組織標本與鏡像方位完全相反。注意二維平面標本像與其三維立體圖像的關系。

12．觀察完一張切片后，應回憶總結一下在該片中辨認了幾種細胞、組織、結構或該器官的特征性結構，每片如此積累，可以大大提高閱片能力和學習效率。

（二）如何觀察和理解切片物像

觀察切片的步驟 應養成由肉眼到低倍，再到高倍，系統觀察標本的習慣。先要了解標本的取材部位、制片方法、切片方位和染色方法，再從宏觀到微觀，由淺入深逐步觀察組織、細胞的微細結構。在掌握組織、細胞光鏡結構的基礎上，可結合觀察某些細胞或結構的電鏡照片，并聯系機能融會貫通深入理解。

正確理解切片的立體形象 人體結構極為復雜，就同一個器官或細胞來說，所切的部位不同或者所切的方向不同，則切片所顯示的物像就不相同。

我們將一個煮熟的雞蛋示意為一個細胞，通過不同方向和部位所作的各種切面，則可得到不同的物像，如Fig.1-2所示。若對呈輻射狀排列的細胞群體作各種切面，其各種物像如Fig.1-3所示。若對呈管狀的器官作各種切面，其形狀如Fig.1-4、Fig.1-5所示；若對呈束狀的器官作各種切面，其形狀如Fig.1-6所示。

切片中的人為現象 在切片中出現的一些人為假象，并非組織結構，應予鑒別：①刀痕：因切片刀鋒有缺口造成組織標本縱行刀痕。②裂紋：組織透明、浸蠟的時間過長，組織脆硬，切片時可引起組織裂開，呈不規則裂紋。在制片過程中，由于組織或細胞各部分結構的收縮不一致，或貼片時水溫過高，也可導致某些人為裂隙。③皺褶：貼片時組織未充分展平。④氣泡：封片時將少許空氣封入切片樹膠中。⑤異留物：如染色時殘留的染料沉渣等。

I. How to use a microscope to examine the slides

Step 1. Locate each part of your microscope according to the above image.

Step 2. Check the glass slides in the box and inform the teacher if there is any one broken or missing.

Step 3. Procedure for examination of the slides using the microscope

A. Gently move the microscope towards you，keeping the distance of about 10 cm between the rim of microscope base and your bench edge.

B. Turn on the power switch，make sure that your light source is functioning，and ensure that the lens nosepiece is rotated properly and the 10X objective lens is fixed.

C. Hold the slide up and examine it first with the naked eye to get an overview of the tissue size，shape and placement on the slide.

D. Put the slide on the stage and fasten it with the stage clip.

E. Look at the stage from the side and turn the coarse focus knob so that the stage goes upward. Move it as close as possible to the objective lens，without touching it！

F. Now，look through the eyepiece，meanwhile turn the coarse knob so that the stage moves away from the objective lens. Continue until the image comes into focus. Then use the fine knob for fine focusing.

G. Move the interesting image in the center of the field of view，then change to the 40X objective lens for further observation.

II. How to examine and understand microscopic images

Tips for examination:

1．Observe the section with naked eyes first for orientation and then at the lower magnification，and increasingly higher magnifications.

2．Be familiar with the sampling site and staining method，which is helpful in understanding the structure.

3．Electron micrograph available in the lab is valuable in connecting the morphology with function of some cells and tissues.

Transition from 3D to 2D:

When viewing a histological sample or specimen，you have to bear in mind that the tissue has been reduced from being three-dimensional to two-dimensional. You are no longer viewing a full specimen or even the exterior of a specimen，but rather a sliver of tissue taken from a specimen.

The following diagrams demonstrate various sectional shapes of a tissue sample cut from different orientations.

Artifacts:

Be aware that each step of tissue preparation introduces artifacts by altering or distorting the natural appearance of cells. Artifacts in slides may result from improper fixation and dehydration，paraffin infiltration，and poor microtome sectioning.

1．Bubbles：The air is sucked in under the coverslip when the mounting media is too thin or dries off.

2．Black precipitates：Formalin-heme pigment forms when the formalin buffer is exhausted and the tissue becomes acidic.

3．Ripples and wrinkles：They can be introduced during cutting and handling of sections when the tissue section stretches unevenly around structures of differing consistencies.

4．Scratches and “chatter”：Scratches are caused by flaws or dirt on the cutting edge, and appear as straight slashes or ragged tears across the specimen.“Chatter” is the visible record of knife vibration. The process of slicing sometimes induces vibrations in the knife edge，which then cause variations in thickness in the section. These appear as narrow parallel bands，usually evenly spaced，across a tissue specimen.