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解像度はどのくらいか？ 視野角はどのくらいか？
本ガイドの対象者 :
顕微鏡カメラを使用している、またはこれから使用したいと考えている研究者の方
本ガイドの内容 :
カメラのスペックと実験との関連性について説明します。
セクション :
仕様の表記について :
| 浜松ホトニクスの表記 | 他メーカーの表記 |
|---|---|
| 有効画素数 | Active pixels, Resolution |
| 画素サイズ | Pixel size |
| 有効素子サイズ | Image area, Active area, Sensor/diagonal |
視野 (Field of view)
カメラの仕様表を見ると、工学的観点から生物学的観点への翻訳だけでなく、カメラメーカーの言語から別の言語への翻訳が必要な場合があります。特に、どの程度の解像度が得られるかを説明する用語はその傾向が顕著です。
まず、有効画素数 (Effective pixel) という言葉について説明します。これは画像を撮影するための画素数のことを示しています。左図のように各画素を拡大すると正方形または長方形のグリッド状になっており、各画素は特定の領域で検出した光子を信号として出力します。
各画素がカバーする領域は、画素のサイズによって異なり、仕様表では画素サイズ (Pixel size) として記載されています。
画素サイズに画素数をかけると、1枚の画像に写る視野が画素数からミリメートルに変換され、有効素子サイズ (Effective sensor size) と表記されます。
画素数を単に画素数ではなく、有効画素数と表現するのはなぜでしょうか？ カメラによっては、センサ上のすべての画素が画像に寄与するのではなく、光電子や電圧を伝達したり保持したりするために使用されるものもあるため、曖昧さを防ぐ表現を使用しています。
分解能
ピクセルサイズの規格は、カメラの分解能にどのように反映されるのでしょうか？
まず、光学系が分解能を制限することを念頭に置くことが重要です。顕微鏡がサンプルを分解できなければ、カメラも画像を見ることができません。そこで、まず顕微鏡の分解能を調べて、分解する対象物の投影像の大きさを算出します。そのためには、分解能を定義する必要があります。
では、分解能はどのように定義されているのでしょうか？
1873年、物理学者エルンスト・アッベ (Ernsr Abbe) は、対物レンズの回折限界をλ/2NAと初めて定義しました。その後、レイリーはこの式を顕微鏡用に改良し、0.61×λ/NA (2つのエアリーパターンを別の物体として見分けるのに必要な距離) としました。
このレイリー方程式から、対物レンズが解像できる最小のものの大きさを算出し、その数値に対物レンズの倍率 (中間光学系がないと仮定) をかけると、そのものがカメラのセンサに映ったときにどれくらいの大きさに見えるかを算出することができます。
この分解能を得るためには、どの程度の画素数が必要なのでしょうか？
光学系の分解能と物体の投影サイズが決まれば、その分解能を満たすために必要な最大許容画素サイズを決定することができます。物体を正確に定義するために必要な最小限の画素数に関する理論的枠組みであるナイキスト (Nyquist) 基準を満たす必要があるため、カメラセンサ上の少なくとも2つの画素に対象物体を確実に投影する必要があります (理想的にはそれ以上、高分解能顕微鏡では2.5～3が推奨されます) 。
例えば、直径約0.7μmのラット肝実質細胞のペルオキシソームを分解しようとする場合を考えてみます。0.7μmの物体を分解するためには、同等以上の分解能を持つ対物レンズが必要です。表1より、最低限必要な倍率は、倍率10倍、NA0.45の対物レンズとなります (もちろん、広い視野を必要としない場合、ほとんどの顕微鏡医は、より視覚的に美しく、情報量の多い画像を得るためにオーバーサンプリングして高倍率を使用します) 。この倍率では投影像が7.5μmになるので、7.5μm÷2＝3.7μm以下の画素サイズのカメラが必要です。
| Table 1. | ||||
|---|---|---|---|---|
| 対物レンズ倍率 | 開口数 (NA) | δ*(=レイリー分解能) (μm) | Δ (=δ x 対物レンズ倍率) (μm) | 適正画素サイズ (μm) |
| 4 | 0.20 | 1.7 | 6.7 | 3.4 |
| 10 | 0.30 | 1.1 | 11.2 | 5.6 |
| 10 | 0.45 | 0.7 | 7.5 | 3.7 |
| 20 | 0.50 | 0.7 | 13.4 | 6.7 |
| 20 | 0.75 | 0.4 | 8.9 | 4.5 |
| 40 | 0.75 | 0.4 | 17.9 | 8.9 |
| 40 | 1.30 | 0.3 | 10.3 | 5.2 |
| 60 | 1.40 | 0.2 | 14.4 | 7.2 |
| 100 | 1.30 | 0.3 | 25.8 | 12.9 |
| 100 | 1.40 | 0.2 | 24.0 | 12.0 |
| *@550 nm | ||||
まとめ :
- カメラが実現できる視野は、各メーカーで有効画素数、有効素子サイズという言葉やそれに準ずるものに反映されています。
- 顕微鏡がサンプルを分解できなければ、カメラも分解できません。
- 顕微鏡が観察対象を分解できると判断した後は、カメラ側が十分な画素数で画像をサンプリングできているかどうかを判断する必要があります。
- カメラがサンプリングの必要性を満たせるかどうかを判断する場合、投影される画像サイズが2ピクセル以上に及ぶ（つまり、3ピクセルの少なくとも一部に触れる）ことを確認します。
- Confirmation
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